Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Clminus -dependent mechanism

Gengru Jiang1, Janet D. Klein1, and W. Charles O'Neill1,2

1 Renal Division, Department of Medicine, and 2 Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Na-K-2Cl cotransporter NKCC1 is an important volume-regulatory transporter that is regulated by cell volume and intracellular Cl-. This regulation appears to be mediated by phosphorylation of NKCC1, although there is evidence for additional, cytoskeletal regulation via myosin light chain (MLC) kinase. NKCC1 is also activated by growth factors and may contribute to cell hypertrophy, but the mechanism is unknown. In aortic endothelial cells, NKCC1 (measured as bumetanide-sensitive 86Rb+ influx) was rapidly stimulated by serum, lysophosphatidic acid, and fibroblast growth factor, with the greatest stimulation by serum. Serum increased bumetanide-sensitive influx significantly more than bumetanide-sensitive efflux (131% vs. 44%), indicating asymmetric stimulation of NKCC1, and produced a 17% increase in cell volume and a 25% increase in Cl- content over 15 min. Stimulation by serum and hypertonic shrinkage were additive, and serum did not increase phosphorylation of NKCC1 or MLC, and did not decrease cellular Cl- content. When cellular Cl- was replaced with methanesulfonate, influx via NKCC1 increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we propose a novel mechanism whereby serum activates NKCC1 by reducing its sensitivity to inhibition by intracellular Cl-. This resetting of the Cl- set point of the transporter enables the cotransporter to produce a hypertrophic volume increase.

Na-K-2Cl cotransport; serum; lysophosphatidic acid; fibroblast growth factor; intracellular chloride; vascular endothelium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE UBIQUITOUS FORM of the Na-K-2Cl cotransporter (NKCC1) has several important functions, including regulation of cell volume and intracellular Cl- concentration ([Cl-]) and the vectorial transport of salt across secretory epithelia. Consequently, the transporter is regulated both by cell volume and intracellular Cl-. The mechanisms responsible for this regulation are complex and poorly understood. Activity of NKCC1 is increased by inhibitors of protein phosphatases and reduced by kinase inhibitors (10, 26), and phosphorylation of the transporter is increased by cell shrinkage or by a reduction in intracellular [Cl-] (6, 9, 16, 17). Phosphorylation occurs at the same site or sites with either stimulus (14), suggesting that NKCC1 is activated by a specific protein kinase. We have recently identified c-jun amino-terminal kinase as a volume-sensitive kinase capable of phosphorylating NKCC1 in vitro (8).

Additional regulation of NKCC1 occurs through myosin light chain (MLC), the phosphorylation of which is increased by cell shrinkage (9, 27). Inhibition of MLC kinase (MLCK) blocks NKCC1 activation in endothelial cells (9), Ehrlich ascites cells (11), colonic epithelial cells (7), and vascular smooth muscle (1), and in endothelial cells this is independent of cotransporter phosphorylation. Activation of the cotransporter in colonic epithelial cells is also affected by agents that alter F-actin (7, 18, 19). The mechanism by which the cellular contractile apparatus or cytoskeleton regulates NKCC1 is not known.

Although intracellular Cl- affects phosphorylation of the cotransporter (6, 17), regulation by this ion is probably more complex and has important implications. Despite being phosphorylated and activated by hypertonic shrinkage, NKCC1 does not produce a net influx of ions, despite being thermodynamically favored (13, 21). Net influx and volume recovery after shrinkage only occur when intracellular [Cl-] is reduced (isosmotic shrinkage). This is consistent with the asymmetric activation of NKCC1 observed in endothelial cells (21). Influx and efflux via NKCC1 are stimulated in parallel by hypertonic shrinkage (increased intracellular [Cl-]), but with isosmotic shrinkage (reduced intracellular [Cl-]) influx is stimulated more than efflux. This has led us to hypothesize that intracellular Cl-, independent of NKCC1 phosphorylation, blocks outward translocation of the unloaded cotransporter, thereby preventing net influx when intracellular [Cl-] is high. Unidirectional influx can still be stimulated but only represents exchange with intracellular ions. Inhibition of NKCC1 by Cl- therefore provides a simple feedback mechanism for regulating cell volume and intracellular [Cl-].

The regulation of NKCC1 by intracellular Cl- becomes important in considering the activation of NKCC1 by growth factors (2, 23, 25). Cotransport-mediated increases in cell volume may occur (22) and may be important for subsequent growth, since inhibition of NKCC1 can block proliferation (5, 24) and overexpression of cotransporters can cause transformation (12). Activation of NKCC1 in the absence of cell shrinkage should lead to an increase in cell volume, but this could be prevented or limited by intracellular Cl-. For instance, direct phosphorylation and activation of NKCC1 in endothelial cells by calyculin does not increase cell volume (10). Thus an increase in cell volume may require either a decrease in intracellular [Cl-] or an increase in the Cl- set point of the cotransporter. Despite the potential importance of NKCC1 in cell growth, the mechanism by which it is activated by growth signals and mediates a hypertrophic volume increase remains unknown.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Endothelial cells were cultured from bovine aortas as previously described in DMEM medium with 10% fetal bovine serum (21). Subconfluent cells were washed twice with serum-free medium (50% DMEM, 50% Ham's F-12, 0.5 mg/ml transferrin, 0.2 mM ascorbate) and then incubated in the same for 72 h with medium changes at 24 and 48 h.

Na-K-2Cl cotransporter activity. Cotransporter activity was measured as unidirectional K+ influx inhibited by 50 µM bumetanide, using 86Rb+ as a tracer, as previously described (21). After a 5-min incubation in assay medium, 86Rb+ was added for 10 min, followed by washing in ice-cold 110 mM MgCl2. Efflux of 86Rb+ was measured as previously described after a 2-h loading period. Medium was removed and replaced at 2-min intervals, and rate coefficients (the fraction of 86Rb+ leaving the cells/min) were calculated before and 5 min after addition of serum. Standard isotonic solution was Earle's salts with HEPES substituted for HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, containing (in mM) 130 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 1 NaH2PO4, 5 glucose, and 26 HEPES, pH 7.4. The measured osmolality was 290 mosmol/l. Hypertonic medium contained an additional 150 mM sucrose so as not to alter the concentration of any transported ion.

Cell volume and Cl- content. Cell water was measured by equilibration of [14C]urea as previously described (21). Cells were incubated with [14C]urea with or without serum or added NaCl for 15 min, followed by rapid washing in ice-cold medium to remove extracellular urea. Medium was made hypertonic by the addition of 75 mM NaCl instead of sucrose, to avoid altering the thermodynamics of the cotransporter. For measurement of Cl- content, cells were preincubated with 36Cl- in standard isotonic medium for 30 min and then washed with ice-cold, isosmotic sodium gluconate 15 min after addition of serum and/or bumetanide.

Phosphorylation assays. These assays were performed as previously described (9), with some modifications. Cells were incubated in phosphate-free DMEM containing 0.1 mCi/ml [32P]orthophosphate for 3 h at 37°C. To determine phosphorylation of NKCC1, cells were extracted with 0.1% SDS in K+-free PBS for 20 min at room temperature. A sixfold volume excess of 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in buffer A [ 250 mM NaCl, 25 mM Tris · HCl, pH 7.5, 100 mM NaF, 10 mM EGTA, 5 mM EDTA, 100 mM sodium pyrophosphate, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 0.5 mM benzamidine] was added, and the samples were sheared with a 26 G needle and incubated on ice for 60 min. After centrifugation to remove insoluble material, supernatants were incubated overnight with monoclonal anti-NKCC1 (T4) and then for 2 h at 4°C with protein G-Sepharose (GIBCO, Grand Island, NY). The Sepharose was washed with buffer A containing 1% Triton X-100 to remove all unbound radioactivity and then suspended in sample buffer (2% SDS, 5% 2-mercaptoethanol, 10% glycerol, in Tris buffer) and boiled. To examine phosphorylation of MLC, cells were extracted with a buffer containing 0.2% Triton X-100, 10 mM PIPES, pH 6.8, 100 mM KCl, 300 mM sucrose, 1 mM EDTA, 1 mM PMSF, 10 mM NaF, and 1 µg/ml leupeptin. The remaining cytoskeletons were solubilized in sample buffer, sheared with a 26 G needle, and boiled. Proteins were separated on 7.5% (NKCC1) or 15% (MLC) SDS polyacrylamide gels, and phosphorylated proteins were identified by autoradiography.

Reagents. 86Rb+, [32P]orthophosphate, [14C]urea, and 36Cl- were purchased from New England Nuclear Life Sciences (Boston, MA). T4 antibody was obtained from the Developmental Studies Hybridoma Bank (University of Iowa). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA) and was heat-inactivated at 60°C for 30 min. All studies were performed with a single lot of serum. Lysophosphatidic acid (LPA) was purchased from Avanti Polar Lipids (Alabaster, AL) and resuspended in standard isotonic medium without calcium and with 0.1% albumin (fatty acid-free) at a concentration of 1 mM. Fibroblast growth factor was a gift from Dr. Robert Swerlick.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The addition of serum to cells maintained for 3 days in a serum-free medium devoid of growth factors produced a rapid stimulation of bumetanide-sensitive Rb+ influx that peaked at 5 min and then declined but remained elevated for at least 20 min (Fig. 1). Maximal stimulation of NKCC1 by serum occurred at 3% (data not shown). LPA and basic fibroblast growth factor also stimulated bumetanide-sensitive Rb+ influx (Fig. 2), but to a lesser extent than serum. Stimulation by LPA occurred at 3 µM and increased up to 100 µM (Fig. 3). Because of the greater stimulation by serum and the solubility problems with LPA, 3% serum was used for all subsequent assays. The stimulation of NKCC1 by serum resulted in an increase in cell volume (Fig. 4). In the absence of serum, inhibition of NKCC1 with bumetanide produced a small, insignificant reduction in cell volume, as previously described (21). Cell volume increased 17% after exposure to serum for 15 min, and almost all of this increase was inhibited by bumetanide. This indicated that stimulation of NKCC1 by serum increases cell volume, ruling out the possibility that serum stimulates NKCC1 through cell shrinkage. Serum also stimulated NKCC1 in hypertonic medium (Fig. 5), indicating that the effects of shrinkage and growth factors are additive. However, serum did not produce a bumetanide-sensitive increase in cell volume in hypertonic medium (data not shown).


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Fig. 1.   Time course of the stimulation of Na-K-Cl cotransporter (NKCC1) by serum. Aortic endothelial cells were preincubated with 3% serum for the times indicated, and then 86Rb+ influx was measured over 10 min in the absence or presence of 50 µM bumetanide. Results are the means of 6 independent assays. Error bars, SE.



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Fig. 2.   Stimulation of NKCC1 by different growth factors. Aortic endothelial cells were preincubated with growth factors for 5 min, and then 86Rb+ influx was measured over 10 min in the absence or presence of 50 µM bumetanide. Results are the means of at least 4 independent assays. Serum, 3% heat-inactivated fetal bovine serum; LPA, 100 µM lysophosphatidic acid; FGF, 100 ng/ml basic fibroblast growth factor. Error bars, SE.



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Fig. 3.   Concentration dependence of the stimulation of NKCC1 by LPA. Aortic endothelial cells were preincubated with LPA for 5 min, and then 86Rb+ influx was measured over 10 min in the absence or presence of 50 µM bumetanide. Results are the means of 7 independent assays. Error bars, SE.



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Fig. 4.   Effect of serum on cell volume. Aortic endothelial cells were incubated with [14C]urea and 3% serum for 15 min in the absence and presence of 50 µM bumetanide as described in MATERIALS AND METHODS. Results are the means of 6 independent assays. Error bars, SE. *P < 0.02 vs. control. **P < 0.01 vs. serum.



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Fig. 5.   Additivity of the effects of serum and hypertonic shrinkage on NKCC1. Aortic endothelial cells were preincubated with 3% serum and/or 150 mM sucrose for 5 min, and then 86Rb+ influx was measured over 10 min in the absence or presence of 50 µM bumetanide. Results are the means of 5 independent assays. Error bars, SE. *P = 0.03 vs. no serum.

Because NKCC1 is a bidirectional cotransporter, the effects of serum on influx and efflux of Rb+ were compared in matched plates of cells (Fig. 6). We have previously used this approach to show that influx and efflux through NKCC1 are equal in endothelial cells under basal conditions and after hypertonic shrinkage (21). Thus, in the present study, no attempt was made to quantitatively compare influx to efflux, which are expressed in different units. However, the stimulation of each by serum can be compared and was threefold greater for influx than for efflux (131 ± 35% vs. 44 ± 17%, P < 0.02), indicating that the activation of NKCC1 by serum is indeed asymmetric.


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Fig. 6.   Comparison of the effect of serum on influx and efflux via NKCC1. Aortic endothelial cells were preincubated with serum for 5 min, after which influx and efflux of 86Rb+ were measured over 5 min in the absence or presence of 50 µM bumetanide. Results are the means of 13 independent assays. Error bars, SE.

To determine how serum activates NKCC1, we examined three known regulatory mechanisms: NKCC1 phosphorylation, MLC phosphorylation, and intracellular [Cl-]. Phosphorylation of NKCC1 was examined by immunoprecipitation from cells preincubated with [32P]orthophosphate. In the autoradiogram shown in Fig. 7, phosphorylated NKCC1 appears as a doublet at ~170 kDa, the intensity of which increased only slightly after addition of serum to serum-starved cells. By comparison, a far greater increase in phosphorylation occurred after hypertonic shrinkage of serum-replete cells. Densitometry performed on 11 separate experiments revealed a 25 ± 11% decrease in NKCC1 phosphorylation after addition of serum. Previous studies in endothelial cells have shown that NKCC1 activity is also regulated by phosphorylation of MLC independent of NKCC1 phosphorylation (9). Phosphorylation of MLC was also assessed in 32P-loaded cells and is apparent as a band at 20 kDa in the autoradiogram shown in Fig. 8. There was a clear increase in phosphorylation after hypertonic shrinkage, as previously described, but no increase after addition of serum to serum-starved cells. Because phosphorylation of NKCC1 and MLC appear to be the principal pathways for activation of NKCC1 by cell shrinkage, these results are consistent with the finding that serum does not produce cell shrinkage. This indicates that serum does not act through a volume-sensitive pathway, which is supported by the fact that stimulation of NKCC1 by hypertonic shrinkage and serum are additive (Fig. 5).


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Fig. 7.   Phosphorylation of NKCC1. NKCC1 was immunoprecipitated from aortic endothelial cells preloaded with [32P]orthophosphate as described in MATERIALS AND METHODS after incubation with 150 mM sucrose or 3% serum for 15 min. NKCC1 appears as a doublet at ~170 kDa (arrow) on this autoradiogram of a 7.5% acrylamide gel.



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Fig. 8.   Phosphorylation of myosin light chain (MLC). Cytoskeletal proteins were extracted from aortic endothelial cells preloaded with [32P]orthophosphate as described in MATERIALS AND METHODS after incubation with 150 mM sucrose or 3% serum for 15 min. MLC appears as a band at 20 kDa (arrow) on this autoradiogram of a 15% acrylamide gel.

To examine the potential role of intracellular Cl-, changes in 36Cl- content were measured in cells preequilibrated with 36Cl (Fig. 9). In the basal state, addition of bumetanide produced a small but significant decrease in Cl- content. The fact that there was not a corresponding decrease in cell volume (Fig. 4) can be explained by the fact that Cl- is a minority anion in cells and is thus altered to a much greater extent by changes in Cl- flux than is cell volume. Addition of serum produced a 25 ± 5% increase in Cl- content, almost all of which was prevented by bumetanide. The increase in Cl- content was greater than the increase in cell water, indicating that intracellular [Cl-] increased. This is consistent with a net influx of Cl- through NKCC1 and indicates that a decrease in intracellular [Cl-] cannot explain the stimulation of NKCC1 by serum.


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Fig. 9.   Effect of serum on cell Cl- content. Cellular 36Cl- was measured as described in MATERIALS AND METHODS with or without 3% serum in the absence and presence of 50 µM bumetanide (bumet). Results are the means of 6 independent assays. Error bars, SE.

Another possible mechanism for stimulation of NKCC1 is a decrease in the sensitivity of the transporter to inhibition by intracellular Cl-. To test this, cells were preincubated in isotonic medium containing methanesulfonate, a cell-permeant anion, in place of Cl- for 30 min. Bumetanide-sensitive influx increased in Cl--depleted cells, consistent with inhibition of NKCC1 by intracellular Cl-, and there was no additional stimulation by serum (Fig. 10). This could not be explained by the possibility that NKCC1 is already maximally stimulated by depletion of Cl-, since further stimulation occurred after hypertonic shrinkage. These results demonstrate that serum stimulates NKCC1 by modulating the inhibitory effect of intracellular Cl-.


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Fig. 10.   Effect of intracellular Cl- depletion on activation of NKCC1. Aortic endothelial cells were preincubated in standard medium or in Cl--free medium containing methanesulfonate for 30 min, after which 86Rb+ influx was measured as described above. A: influx in the absence or presence of 3% serum. Results are the means of 5 independent assays. Error bars, SE. B: influx in the absence or presence of 150 mM sucrose. Results are the means of 3 independent assays. Error bars, SE. *P < 0.01, **P < 0.05 vs. isotonic.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Investigation of the regulation of NKCC1 has focused primarily on cell volume and intracellular Cl-. Although these parameters are linked under physiological conditions, they are clearly independent regulators of NKCC1. A decrease in cell volume can increase NKCC1, even in the face of an increase in intracellular [Cl-] (i.e., hypertonic shrinkage), and a decrease in intracellular [Cl-] can stimulate NKCC1 in swollen cells (i.e., hypotonic swelling). Our results demonstrate that growth factors are an additional, independent stimulus for NKCC1, since neither cell volume nor intracellular [Cl-] was decreased. Furthermore, this stimulation is independent of NKCC1 phosphorylation, which has been thought to be the principal mechanism for regulation by both cell volume and intracellular Cl- (6, 14, 16, 17), and independent of MLC phosphorylation, an additional volume-sensitive regulatory pathway (1, 9). The fact that the stimulation by serum was abolished by removing intracellular Cl- points instead to modulation of the Cl- sensitivity of NKCC1 that is independent of phosphorylation.

This novel mechanism for regulation of NKCC1 enables cells to respond appropriately to growth stimulation. While regulation of NKCC1 by cell volume and intracellular Cl- is homeostatic, maintaining cell volume and intracellular [Cl-], activation by growth factors is independent of either and is therefore dynamic, with the potential to increase cell volume and intracellular [Cl-]. This potential is only realized because the normal feedback inhibition that regulates intracellular [Cl-] (and therefore cell volume as well) is abrogated, thus explaining the hypertrophic volume increase that is observed. A rapid increase in cell volume is an early response to growth stimuli and may be required for normal growth (20). Inhibition of NKCC1 by either furosemide or bumetanide blocks the proliferative response of endothelial cells to fibroblast growth factor (24).

Although changes in intracellular [Cl-] can alter phosphorylation of NKCC1, some previous data have supported, albeit indirectly, an additional effect of Cl- on NKCC1. Of particular interest are the differing effects on NKCC1 of hypertonic and isosmotic shrinkage, between which the only major difference is the intracellular [Cl-] (increasing in the former and decreasing in the latter). Although NKCC1 phosphorylation and unidirectional influx increase, and net influx is thermodynamically favored in each case, net influx of ions only occurs after the latter (21). This suggests an effect of Cl- on the kinetics of NKCC1 (for review, see Ref. 20). Further evidence for this is the trans effect of intracellular anions on the Na-K-Cl cotransporter in internally perfused squid axons (3, 4), although an effect on cotransporter phosphorylation cannot be ruled out in these studies. One simple explanation is that Cl-, either directly or indirectly, blocks translocation of the empty cotransporter to the outer aspect of the plasma membrane, preventing net influx and permitting only ion exchange, thereby providing a simple, negative-feedback regulation of intracellular [Cl-]. Removal of this block would produce an asymmetric stimulation of influx and an increase in cell volume and Cl- content, as was observed. However, this cannot be the only mechanism, since efflux also increased, and the increase in unidirectional influx by serum in hypertonic medium was not accompanied by any net influx.

The mechanism by which serum alters the Cl- sensitivity of NKCC1 is unclear, in part because the Cl--sensing pathway is poorly understood. Although many effects of growth factors are mediated through phosphorylation, our data do not support changes in the phosphorylation of NKCC1. However, we cannot rule out the possibility that there is a change in phosphorylation sites without a change in overall phosphorylation. If intracellular Cl- affects NKCC1 through direct binding, it is difficult to envision how this could be altered by serum in the absence of NKCC1 phosphorylation. Alternatively, NKCC1 may be regulated by a Cl--sensing protein whose properties are regulated by growth factor-dependent phosphorylation.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-47994.


    FOOTNOTES

Address for reprint requests and other correspondence: W. C. O'Neill, Emory Univ., Renal Division WMB 338, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: woneill{at}emory.edu).

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.

Received 11 January 2001; accepted in final form 27 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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