A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells

Christian Lytle

Division of Biomedical Sciences, University of California, Riverside, California 92521

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

When Na-K-2Cl cotransport is activated in duck red blood cells by either osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, phosphorylation of the transporter occurs at a common set of serine/threonine sites. To examine the kinetics and regulation of the activating kinase, phosphatase activity was inhibited abruptly with calyculin A and the subsequent changes in transporter phosphorylation and activity were determined. Increases in fractional incorporation of 32P into the transporter and uptake of 86Rb by the cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in this manner, the activating kinase was 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine or fluoride. The inhibitory effect of swelling on kinase activity was progressively relieved by calyculin A, suggesting that the kinase itself is switched on by phosphorylation. The kinetics of activation by calyculin A conformed to an autocatalytic model in which the volume-sensitive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).

cell volume regulation; calyculin A; N-ethylmaleimide; staurosporine

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

ANIMAL CELLS CONTROL THEIR volume by modulating membrane transporters and metabolic enzymes that remove or add cytoplasmic solutes (6, 24, 29, 33, 40). For example, osmotic shrinkage of duck red blood cells induces a net uptake of solute via Na-K-2Cl cotransport, whereas cell swelling results in solute loss via K-Cl cotransport (12, 19). How the volume signal is detected and transduced is incompletely understood.

It has been postulated that alterations in cell volume might be sensed as changes in membrane tension, cytoskeletal architecture, or cytoplasmic macromolecular crowding (25, 26, 33, 39, 45). Demonstrations that changes in cytoplasmic protein concentration independent of cell size activate volume-responsive transporters in red blood cells (5, 33), as well as channels in barnacle muscle cells (46), support the macromolecular crowding hypothesis of volume perception.

Transduction of the volume signal appears to involve phosphorylation. In red blood cells from many species, agents that inhibit serine/threonine protein phosphatases (calyculin A, endothall thioanhydride, and okadaic acid) and kinases (staurosporine) mimic the effect on volume-regulatory transporters of osmotic shrinkage and swelling, respectively (30). Moreover, in various cell systems, shrinkage-induced activation of Na-K-2Cl cotransport is associated with direct phosphorylation of the transport protein itself (10, 17, 22, 28).

In addition to osmotic shrinkage, cotransport in duck red blood cells can also be activated by norepinephrine (via cAMP), fluoride, or calyculin A, each of which promotes phosphorylation of the same set of serine and threonine sites (21). These results suggest that the transporter is switched on by a single protein kinase and switched off by a type 1 protein phosphatase. Whether cell shrinkage, norepinephrine, and fluoride activate by stimulating phosphorylation, inhibiting dephosphorylation, or both, remains unsettled. To gain insight into the activation process and to determine whether the various activating factors (cell shrinkage in particular) exert their effects through the kinase, we investigated the kinetics of transporter phosphorylation after inhibition of dephosphorylation with calyculin A.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials. Analytic grade chemicals were from Fisher Scientific. Bumetanide, ouabain, and N-ethylmaleimide (NEM) were from Sigma. 86RbCl was from DuPont NEN. Calyculin A (LC Laboratories) was prepared as a 250 µM stock solution in ethanol. Endothall thioanhydride (LC Laboratories) was dissolved in DMSO to a final concentration of 250 mM. Staurosporine (Molecular Probes) was dissolved in DMSO to a final concentration of 30 mM.

Preparation of red blood cells. Blood was drawn into heparinized syringes from the brachial vein of female White Pekin ducks (Anas platyrynchos). After centrifugation, the plasma and buffy coat were discarded and the red blood cells were washed three times in ice-cold duck fluxing solution (DFS; 146 mM NaCl, 6 mM KCl, 0.1 mM Na2PO4, 10 mM glucose, 20 mg/l penicillin, 45 mg/l streptomycin, and 20 mM Na-TES, pH 7.40 at 41°C, 320 mosmol/kgH2O). Before each experiment, cells were suspended in DFS (hematocrit 2.5%) and preincubated for 45 min at 41°C in a gyrating water bath to achieve steady states with respect to ion and water contents.

In experiments with the various anisotonic media, cells were also preincubated 3-10 min (hematocrit 5%; see Figs. 1-8). To obtain better temporal resolution of the activation process, all incubations were carried out at 30°C, which is 11°C cooler than the core body temperature of the duck. To render media hypertonic (425 mosmol/kgH2O), sucrose was added. A hypotonic (220 mosmol/kgH2O) incubation solution was achieved by lowering the concentration of NaCl. All preincubation and flux media also contained 50 µM ouabain to eliminate Na-K pump activity, which tends to confuse interpretation of flux data when passive transport processes are examined.

Transport assays. Na-K-2Cl cotransport activity was estimated using 86Rb as a surrogate for K, as previously described (21). Uptake was initiated by the addition of isotope and terminated after 15-60 s by prompt addition to each vessel of ice-cold "stop solution" (DFS containing 250 µM bumetanide). Over this brief flux interval, the rate of uptake of 86Rb and cell volume were essentially constant. To remove extracellular isotope, cells in each vessel were washed three times in the stop solution. 86Rb was assayed by gamma ray spectroscopy (Beckman). Influx rates were calculated from intracellular 86Rb content, extracellular specific activity, initial hematocrit, and duration of the flux. The component of 86Rb influx inhibited by 100 µM bumetanide was attributed to Na-K-2Cl cotransport; this component constituted ~95% of the ouabain-insensitive 86Rb influx in osmotically shrunken cells. Bumetanide-sensitive 86Rb influx is expressed in millimoles per liter original cells per minute. In these experiments, the contribution of swelling-induced K-Cl cotransport to bumetanide-sensitive 86Rb influx was negligible.

Activation rate. To determine the time course of activation following stimulation, a series of short (0.3-3 min) 86Rb influx assays were performed in rapid succession. The bumetanide-sensitive rate was plotted against the midpoint of the sampling interval.

Phosphorylation. The phosphorylation state of the Na-K-2Cl cotransport protein was measured as previously described (21). Briefly, cells were incubated at 41°C for 3 h in DFS containing 150 µCi/ml [32P]orthophosphate, washed once in ice-cold DFS, and then reincubated at 30°C in a medium containing the cotransport activator along with 50 µM ouabain. Samples were centrifuged (10 s, 6,000 g) and flash frozen in liquid nitrogen. The cotransport protein was immunoprecipitated selectively from detergent extracts using the monoclonal antibody T14 (23) and then separated by gel electrophoresis. The 32P content of the 146-kDa transporter band was quantified by autoradiography using a Molecular Dynamics phosphoimager.

Data analysis. Activation time course data were fit to kinetic models by nonlinear least squares iteration using Deltapoint Deltagraph or MicroMath Scientist software. Two schemes were tested under conditions in which dephosphorylation is inhibited by calyculin A. In this circumstance, model A (see Fig. 9) takes the form

T <LIM><OP><ARROW>→</ARROW></OP><UL><AR><R><C></C></R><R><C><IT>k</IT><SUB>2</SUB> ⋅ VK</C></R><R><C></C></R></AR><AR><R><C>SK</C></R><R><C>↓</C></R><R><C><IT>k</IT><SUB>1</SUB> ⋅ SK<SUB>P</SUB></C></R></AR></UL></LIM> T<SUB>p</SUB>


where T represents the transporter, SK is the kinase that activates the transporter, VK is a volume-sensitive kinase that activates SK, and TP and SKP denote the phosphorylated states of T and SK. The conversion of ATP to ADP in each phosphorylation reaction is omitted for simplicity. In this scheme, the rate of transporter phosphorylation (T right-arrow TP) depends on the number of active kinase units (SKP) and their intrinsic activity (k1). At a given cell volume, VK catalyzes the conversion SK right-arrow SKP at a particular rate (k2). The irreversible phosphorylation of T is described by the differential equation
<FR><NU>dT<SUB>P</SUB></NU><DE>d<IT>t</IT></DE></FR> = <IT>k</IT><SUB>1</SUB>SK<SUB>P</SUB>(1 − T) (1)
in which SKP is a function of time (t)
SK<SUB>P</SUB> = SK<SUB>t</SUB>(1 − <IT>e</IT><SUP>(−<IT>k</IT><SUB>2</SUB><IT>t</IT>)</SUP>) (2)
where SKt represents the sum of SK and SKP. Hence, as SK is converted to SKP by VK, the rate constant for transporter phosphorylation increases. Activation time course data were fit directly to this differential equation by numerical techniques using MicroMath Scientist software.

Model B differs only in the way SKP is formed (Eq. 2). This model assumes that a product of the SKP reaction acts as a catalyst for further SKP formation. Let SKP(t) equal the initial amount of SKP [SKP(0)] plus the amount gained by the reaction at time t, and SKtotal represent the sum of SK(t) and SKP(t). The model assumes that the rate of SKP formation will be proportional to SK(t) and SKP(t) as described by the differential equation
<FR><NU>dSK<SUB>P(<IT>t</IT>)</SUB></NU><DE>d<IT>t</IT></DE></FR> = <IT>k</IT>[SK<SUB>P(<IT>t</IT>)</SUB>][SK<SUB>(<IT>t</IT>)</SUB>] (3)
whose general solution is
SK<SUB>P(<IT>t</IT>)</SUB> = SK<SUB>P(0)</SUB> + <FR><NU>SK<SUB>total</SUB></NU><DE>1 + <IT>re</IT><SUP>SK<SUB>total</SUB>(−<IT>kt</IT>)</SUP></DE></FR> (4)
where r and k are constants. Equation 4 is a logistic function analogous to those used previously to model autocatalytic reactions such as the activation of trypsinogen and the activation of protein kinases by autophosphorylation (1, 9). Activation time course data were fit to Eq. 4 with the aid of Deltapoint Deltagraph software.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Figure 1A shows changes in Na-K-2Cl cotransport in duck red blood cells and the concurrent phosphorylation of the cotransport protein following activation by a step decrease in cell volume at 30°C. The transition from the resting to the active phosphorylated state could be fit with a single exponential function (Fig. 1A). Because this process (half time, 1.3 min) is >50 times slower than osmotically induced changes in red blood cell volume (27), it must reflect a rate-limiting event in either cell volume perception or signal transduction. Changes in 86Rb transport coincided closely with 32P incorporation. Previous measurements of phosphorylation stoichiometry (21) have suggested that each cotransporter acquires about five phosphates on going from an inactive state (swollen cells) to a transporting state (shrunken cells).


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Fig. 1.   Activation time course of Na-K-2Cl (NKCC) cotransport and phosphorylation of cotransport protein following cell shrinkage. Cells were preincubated for 3 min in isotonic (A) or hypotonic (preswollen; B) duck fluxing solution (DFS) and then transferred for shrinkage to hypertonic DFS containing ouabain. Aliquots were removed every 20 s and exposed for 20 s to 86Rb with or without bumetanide. Transport activity (bumetanide-sensitive 86Rb influx rate) was plotted against midpoints of consecutive influx intervals (bullet ). Curve in A represents a single exponential fit of data yielding a rate constant of 0.52 min-1. Concurrent changes in transporter phosphorylation (open circle ) were measured on paired 32P-labeled cells as described in EXPERIMENTAL PROCEDURES. Ordinate scales were adjusted so that maximum activity corresponds to maximum phosphorylation. Curve through 32P data (B) was drawn by eye. Incubations were conducted at 30°C. One of three similar experiments is shown.

The kinetics of this transition depends on the volume of the cell before shrinkage. Hyposmotic swelling for 3 min before hyperosmotic shrinkage (Fig. 1B) delayed the onset of both activation and phosphorylation. No such delay was observed in cells subjected to preswelling for only 15 s at 30°C or for as long as 5 min at 2°C (data not shown), indicating that the effect of swelling depends on both temperature and time. At the temperature employed in all subsequent experiments in this series (30°C), preswelling for >3 min produced no greater delay in activation. Ten seconds after exposure to hypertonicity, both preswollen cells and control cells shrank to the same level of cell water (1.2 l/kg cell solid), which excludes the possibility that the brief preswelling delays or blunts subsequent cell shrinkage. These results suggest that transporter phosphorylation depends on an intermediate that forms in shrunken cells and breaks down in swollen cells.

Because the level of phosphorylation reflects a competition between simultaneous kinase and phosphatase activities (21, 36), shrinkage could act by stimulating the kinase, suppressing the phosphatase, or both. To determine whether shrinkage stimulates the kinase, the rates of phosphorylation after addition of calyculin A were compared in swollen and shrunken cells. This approach takes advantage of the fact that calyculin A enters the duck red blood cell within seconds and blocks ongoing dephosphorylation of the transporter at all volume-sensitive sites (21). The time courses of transporter activation and phosphorylation in response to calyculin A are shown under hypotonic and hypertonic conditions in Fig. 2. In shrunken cells, phosphorylation commenced after a very brief delay and progressed quickly to a steady plateau, whereas in swollen cells it was delayed for several minutes and increased less rapidly. Thus cell shrinkage hastens both the onset and rate of phosphorylation. Fractional changes in 86Rb transport and 32P incorporation (Figs. 1 and 2) were roughly comparable. Similar volume-sensitive responses (data not shown) were obtained with endothall thioanhydride, another potent inhibitor of type 1 and type 2A protein phosphatases (8).


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Fig. 2.   Time course of Na-K-2Cl cotransporter activation and phosphorylation after inhibition of dephosphorylation with calyculin A. Cells were swollen for 3 min by incubation in hypotonic DFS and then transferred to hypertonic (A; shrunken cells) or hypotonic (B; swollen cells) DFS containing calyculin A and ouabain. Aliquots were removed every 30 s and exposed for 30 s to 86Rb with or without bumetanide. Transport activity (bumetanide-sensitive 86Rb influx rate) was plotted against midpoints of consecutive influx intervals (bullet ). Solid and dashed curves represent fits of 86Rb influx data to a logistic function and to a volume-sensitive kinase model, respectively (see DISCUSSION). Concurrent changes in transporter phosphorylation (open circle ) were measured on paired 32P-labeled cells as described in EXPERIMENTAL PROCEDURES. Ordinate scales were adjusted to equalize maximal activity and maximal phosphorylation. A and B depict separate experiments. Incubations were conducted at 30°C. One of three similar experiments is shown.

Changes in cell volume did not alter the final extent or pattern of transporter phosphorylation elicited by calyculin A (21), only the rate at which this state was reached (Fig. 2). Similar results were obtained in measurements of transport activity; decreases in cell volume hastened the onset and rate of activation by calyculin A but did not alter the final state of activation (Fig. 3). After reaching this state, transport activity was unaffected by subsequent osmotic perturbations (data not shown). Hence, once the transporter is stably phosphorylated, its operation becomes insensitive to changes in cell volume.


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Fig. 3.   Cell swelling delays activation of Na-K-2Cl cotransporter by calyculin A. Cells were swollen in 220 mosmol/kgH2O DFS for 3 min and then transferred to hypertonic DFS (shrunken cells), isotonic DFS (control), or hypotonic DFS (swollen cells), each containing calyculin A. Aliquots were removed every 0.5-3.0 min and exposed for 0.5-3.0 min to 86Rb with or without bumetanide. Transporter activity (bumetanide-sensitive 86Rb influx rate) was plotted against midpoints of consecutive influx intervals. Final steady-state rates were measured between 21 and 23 min. Solid and dashed curves represent fits of 86Rb influx data to a logistic function and to a volume-sensitive kinase model, respectively (see DISCUSSION). One of two similar experiments is shown.

The rate of phosphorylation in cells treated with calyculin A depended not only on cell volume but also on time. In Figs. 2 and 3, the progressive increase in the slope of the curve indicates that phosphorylation (activation) accelerates over time in the presence of calyculin A. The kinetics of activation conformed closely to an autocatalytic model described by a logistic function (model B; see DISCUSSION). Less satisfactory fits were obtained with a kinetic model in which the rate of activation accelerates monoexponentially (model A; see DISCUSSION).

An investigation of the early volume-sensitive phase of the calyculin response is shown in Fig. 4. The initial rate of activation, presumed to reflect kinase activity, was rapid in cells of normal volume (water content, 1.52 l/kg cell solid). A rise in water content of only 7%, to 1.63 l/kg cell solid, resulted in a 70% decrease in activation rate. The initial rate of phosphorylation exhibited a similar sensitivity to cell volume (Fig. 4B). A plot of these initial rates against cell water content shows that activation (Fig. 5A) and phosphorylation (Fig. 5B) are similarly inhibited by cell swelling and stimulated by cell shrinkage.


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Fig. 4.   Effect of cell volume on initial rate of Na-K-2Cl cotransporter activation and phosphorylation by calyculin A. A: cells were preincubated at 30°C in DFS of different osmolality for 5 min and then exposed to calyculin A. Transporter activity (bumetanide-sensitive 86Rb influx rate) was measured every 15 s in 7 successive 15-s influx assays. Cell volume (water content) was determined on samples removed after 1 min; values in l/kg cell solid are noted adjacent to each trace. * Control (isotonic) water content. B: time course of transporter phosphorylation in 32P-labeled cells. A and B represent separate experiments.


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Fig. 5.   Volume dependence of kinase activity. Initial linear rise in Na-K-2Cl cotransporter activity (A) or phosphorylation (B) after phosphatase inhibition is plotted as a function of cell water content. Filled symbols denote experiments in which cells were preincubated for 5 min in anisotonic media to allow effect of cell swelling to develop fully before adding calyculin A. Open symbols denote experiments in which cells were exposed to anisotonicity and calyculin A simultaneously. Shaded bars indicate normal steady-state cell water content (1.55 l/kg cell solid). All incubations were performed at 30°C.

Fluoride and norepinephrine can also activate the transporter (31) and promote its phosphorylation at similar sites (21). These agents might act by stimulating the kinase, like cell shrinkage, or by inhibiting the phosphatase, like calyculin A. To distinguish between these possibilities, the effects of the two agents on the rate of activation after addition of calyculin A were determined. Fluoride (Fig. 6) and norepinephrine (Fig. 7) had no significant effect on the time course of activation in either shrunken or swollen cells. In control cells (without calyculin A), both fluoride and norepinephrine evoked near maximal transport within 5 min, and this response was blocked by cell swelling (data not shown). As reported previously (21), all these modes of stimulation (shrinkage, norepinephrine, fluoride, and calyculin A) were blocked by staurosporine (Figs. 6, 7, and 8) with a similar IC50 (0.7 µM), consistent with the idea that each signal is transduced via the same staurosporine-sensitive kinase. Because fluoride and norepinephrine do not affect the process of phosphorylation, they must stimulate the transporter by inhibiting its dephosphorylation.


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Fig. 6.   Activation of Na-K-2Cl cotransporter by calyculin A is not affected by fluoride. Cells were preswollen in hypotonic DFS for 5 min and then transferred to hypotonic or isotonic DFS containing calyculin A. Open symbols, no fluoride; filled symbols, medium contained 10 mM fluoride; black-triangle, medium also contained 10 µM staurosporine (stauro). Changes in cotransport were measured as in Figs. 2-4. All incubations were performed at 30°C. A control assay confirmed that fluoride stimulated cotransport in absence of calyculin A (not shown). Similar results were obtained in 2 experiments.


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Fig. 7.   Activation of Na-K-2Cl cotransporter by calyculin A is not affected by norepinephrine (NE). Onset of activation was measured as in Fig. 6 except that 10 µM NE replaced fluoride. Open symbols, no NE; filled symbols, medium contained 10 µM NE; black-triangle, medium also contained 10 µM staurosporine. One of three similar experiments is shown.


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Fig. 8.   N-ethylmaleimide (NEM) inhibits Na-K-2Cl cotransporter phosphorylation. A: 32P-labeled cells were prestimulated by incubation (7% hematocrit, 41°C, 5 min) in DFS containing 100 mM sucrose (hypertonic; shrinkage stimulated) or 0.2 µM calyculin A. In one case (square ), hypertonic DFS was supplemented with 5 µM staurosporine. NEM was then added from a freshly prepared 0.5 M stock solution in DMSO to yield indicated concentration. Ten minutes later, cells were abruptly frozen in liquid nitrogen. Level of transporter phosphorylation was determined as described in EXPERIMENTAL PROCEDURES. Data are means ± range of duplicate measurements. B: cells were preincubated (7% hematocrit, 30°C, 8 min) with or without 750 µM NEM and then treated with 0.2 µM calyculin A. Aliquots were removed every 30 s and exposed for 30 s to 86Rb with or without bumetanide. Cotransport activity (bumetanide-sensitive 86Rb influx rate) was plotted against midpoints of consecutive influx intervals. One of three similar experiments is shown.

Treatment of duck red blood cells with NEM, a cell-permeant sulfhydryl agent, inhibits Na-K-2Cl cotransport and activates KCl cotransport (30). As shown in Fig. 8, NEM inhibited the volume-sensitive kinase. Treatment of shrunken duck red blood cells with 750 µM NEM lowered phosphorylation to the level seen in the presence of staurosporine alone (Fig. 8A) and completely inhibited Na-K-2Cl cotransport (Fig. 8B), but this concentration had little effect on cells previously exposed to calyculin A. Thus the effects of NEM resemble those of staurosporine and cell swelling. If NEM promotes dephosphorylation by inhibiting the kinase, it should also block activation by calyculin A, as indeed it did (Fig. 8B). In cells treated with NEM, as in cells treated with staurosporine, transport activity was near zero and remained unresponsive to calyculin A in both normal (Fig. 8B) and swollen cells (not shown). Hence, the inhibitory effect of NEM and staurosporine, unlike that of cell swelling, is not relieved gradually by calyculin A.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

It is generally believed that Na-K-2Cl cotransport is turned on when the transporter is phosphorylated at multiple serine and threonine residues (10, 17, 22, 28). In the duck red blood cell, four activating stimuli (osmotic shrinkage, norepinephrine, fluoride, and calyculin A) evoke phosphorylation at cognate sites, implicating a common protein kinase (21). To investigate the activation kinetics of this putative enzyme, we treated cells with calyculin A to inhibit the protein phosphatase opposing the kinase and measured the rate at which the transporter becomes active. For comparison, the rate of phosphorylation of the cotransport protein was measured under the same conditions. Observed in this manner, the activating kinase was found to be 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or NEM, and 3) unaffected by the presence of norepinephrine or fluoride. Fractional incorporation of 32P by the protein was closely correlated with transport activity. Thus the transporter is switched on by a protein kinase that is modulated by cell volume (referred to herein as CT-kinase) and switched off by a protein phosphatase that is inhibited by cAMP, fluoride, calyculin A, or endothall thioanhydride (PP).

The concept of a volume-sensitive kinase first emerged from the work of Jennings and Al-Rohil (15) on swelling-activated K-Cl cotransport in rabbit red blood cells. These investigators observed a significant delay between swelling and the appearance of K-Cl cotransport but no lag in deactivation after volume was restored to normal. Their interpretation based on relaxation kinetics suggested a simple model in which the K-Cl cotransporter (or a protein that activates it) converts between two states, inactive when phosphorylated and active when dephosphorylated, depending on the relative activities of a volume-sensitive protein kinase and a type 1 protein phosphatase. The involvement of a swelling-inhibited kinase was supported by two key observations: 1) agents that inhibit protein phosphatases (fluoride, vanadate, inorganic phosphate, and okadaic acid) prevented swelling activation of K-Cl cotransport or delayed activation at submaximal concentrations, and 2) swelling activation of K-Cl cotransport is the result of slower deactivation (by a kinase) rather than faster activation (by a phosphatase) (15, 16).

Our results are consistent with those obtained previously in studies on other cells. Klein et al. (18) found that osmotic swelling of aortic endothelial cells delayed the activation of Na-K-2Cl cotransport by calyculin A and proposed that some critical phosphorylation step is affected by cell volume. In lymphocytes, activation of the Na/H exchanger after phosphatase inhibition with okadaic acid was hastened by cell shrinkage (2).

Once PP is inhibited by calyculin A, dephosphorylation of the Na-K-2Cl cotransporter is prevented, and transport activity at some point thereafter becomes insensitive to changes in cell volume. Thus all the effects of shrinkage and swelling on transport activity appear to be exerted through the reactions that determine transporter phosphorylation. Cell volume clearly modulates CT-kinase, but whether it also modulates PP remains uncertain. There is evidence that cell shrinkage slows the rate of decrease of Na-K-2Cl cotransport following inhibition of protein kinases with either K252a or Mg/ATP depletion (32). Thus shrinkage might promote transporter phosphorylation in two ways: by stimulating CT-kinase and by inhibiting PP.

Two lines of evidence suggest that CT kinase is itself activated by phosphorylation. First, the inhibitory effect of cell swelling on transporter phosphorylation develops and reverses in a time- and temperature-dependent manner. This implies that CT-kinase activity requires something that is formed on cell shrinkage and broken down on cell swelling. Second, when cells are treated with potent and specific inhibitors of type 1 and type 2A serine-threonine protein phosphatases, namely calyculin A or endothall thioanhydride (8, 14), CT-kinase activity increases progressively at a rate that depends on cell volume. In swollen cells, phosphorylation proceeds slowly at first but progressively accelerates to a moderately fast rate (Figs. 2 and 3). Because the inhibitory effect of swelling on CT-kinase activity is gradually overcome by phosphatase inhibition, it follows that CT-kinase is regulated by reversible phosphorylation at serine-threonine residues.

Two hypothetical models of CT-kinase regulation are illustrated in Fig. 9. Both assume that the enzyme becomes active when phosphorylated at sites susceptible to dephosphorylation by a calyculin-sensitive protein phosphatase (PP). In model A (Fig. 9), activation of cotransport results from a catalytic cascade in which a shrinkage-enhanced kinase (VS-kinase) phosphorylates CT-kinase, which in turn phosphorylates the cotransporter, leading to increased cotransport. This cascade is an attractive mechanism for volume signal transduction, as it would allow for signal amplification, feedback, cross talk, and branching (13). The model intuitively accounts for increased cotransporter activity in the presence of calyculin A and its complex dependence on cell volume. By inhibiting PP, calyculin A allows both VS-kinase and CT-kinase to operate unopposed, resulting in an accumulation of phosphorylated CT-kinases and phosphorylated cotransporters. Swelling cells inhibits VS-kinase. Consequently, CT-kinase activity is decreased, but cotransporter phosphorylation gradually increases as additional CT-kinase units are brought on line by the action of the residual VS-kinase. In shrunken cells, in which VS-kinase is stimulated, CT-kinase is most active, and cotransporter phosphorylation progresses rapidly to completion. Although this scheme is superficially attractive, fits of the data to the model systematically underestimate the sigmoidal character of the activation curve at each cell volume tested (Figs. 2 and 3)


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Fig. 9.   Hypothetical schemes for activation of Na-K-2Cl cotransporter by cell shrinkage and calyculin A. Transporter moves ions when phosphorylated at multiple serine and threonine residues. This phosphorylation is determined by relative activities of an unidentified protein kinase (CT-kinase) and a calyculin-sensitive protein phosphatase (PP). In model A (A), CT-kinase becomes active when phosphorylated by another kinase (VS-kinase) at sites that are dephosphorylated by PP. VS-kinase is stimulated by cell shrinkage and inhibited by cell swelling. Model B (B) illustrates an alternative scheme in which CT-kinase phosphorylates itself in addition to transporter; this autophosphorylation partially relieves inhibitory effect of cell swelling (or potentiates stimulatory effect of cell shrinkage). In both models, restoration of cell volume causes phosphorylation processes to subside, allowing PP to outpace them. Fits of activation time course data to each model favor model B. See text for details.

Better fits were obtained using a logistic model analogous to those employed to analyze autocatalytic reactions (1, 9). The essential element of this model is a positive feedback loop in which CT-kinase is stimulated, not only by cell shrinkage, but also autocatalytically by a product of its own reaction. The stimulatory product could be the phosphorylated form of the transporter, another target protein, or CT-kinase itself via an autophosphorylation mechanism. This autophosphorylation scheme is presented as model B in Fig. 9. In this case, autophosphorylation of CT-kinase would relieve the inhibitory effect of swelling or potentiate the stimulatory effect of shrinkage. The proposed action of autophosphorylation in stabilizing the active conformation of CT-kinase is analogous to a similar mechanism in Ca/calmodulin-dependent protein kinase II (41). As shown in Figs. 2 and 3, this positive feedback model provides excellent fits to the activation time course data at each cell volume tested.

Because calyculin A elicits rapid net phosphorylation of the cotransporter when added to unperturbed cells (Figs. 4 and 5), the active form of CT-kinase must be present at moderate levels in cells of normal volume. Volume deviations of only 5% are sufficient to produce disproportionately large (~50%) changes in the initial rate of activation by calyculin A (Fig. 5). Because CT-kinase activity is reciprocally related to increases and decreases in normal cell volume, it is in a position to affect both shrinkage-induced and swelling-induced processes. Although it has been argued that a single kinase model could not allow for a resting volume at which both shrinkage- and swelling-induced transporters are quiescent (7, 40), the argument overlooks the possibility that at any particular cell volume CT-kinase might act on different transporters at different rates or that the action of CT-kinase might be opposed by different phosphatases.

Whereas calyculin A makes red blood cells behave as if shrunken, sulfhydryl-alkylating agents like NEM make them behave as though they are swollen, i.e., Na-K-2Cl cotransport is inhibited and K-Cl cotransport is activated (20, 30). However, NEM is without effect on either transporter in cells previously exposed to calyculin A, and calyculin A is without effect on either transporter in cells previously exposed to NEM (16, 43) (Fig. 8). Thus NEM apparently inhibits a kinase. The finding that higher concentrations of NEM (>750 µM) irreversibly attenuated Na-K-2Cl cotransport in calyculin-treated cells suggests that the transport protein also harbors critical thiols, consistent with the observation that NEM labels the Na-K-2Cl transporter in a bumetanide-protectable fashion (37).

Like NEM, staurosporine makes red blood cells behave as though swollen (3). In sheep red blood cells, the stimulatory effect of staurosporine on K-Cl cotransport was half-maximal at 0.4 µM (3), comparable to the concentration producing half-maximal inhibition of the Na-K-2Cl cotransporter in duck red blood cells (~0.7 µM; Ref. 21). Because staurosporine prevents phosphorylation of the Na-K-2Cl cotransporter in the presence of calyculin A (Figs. 6-8; Ref. 21), it appears to inhibit the activating kinase (CT-kinase), rather than stimulate the deactivating phosphatase (PP). This finding is consistent with the ability of this agent to inhibit a variety of protein kinases (38).

When incubated in native plasma or an isotonic artificial medium, duck red blood cells assume a "set point volume" at which both shrinkage-induced (Na-K-2Cl) and swelling-induced (K-Cl) cotransport processes are nearly quiescent (11). A shift away from this set point volume arouses one transporter and further suppresses the other. The set point volume is modulated by a variety of internal and external influences. Norepinephrine, cAMP, cytoplasmic magnesium, hypoxia, and calyculin A all raise the set point volume and thereby cause Na-K-2Cl cotransporters to become active at normal cell volume (30, 42, 44). A similar mechanism appears to underlie the coordination of shrinkage-activated Na/H exchangers and swelling-activated K-Cl cotransporters in dog red blood cells (33, 34). These results have led to the proposal that swelling- and shrinkage-induced transporters are orchestrated by a common mechanism (6, 33-35). That common mechanism could be CT-kinase. Indeed, the kinase that deactivates K-Cl cotransport shares several characteristics with the kinase that activates Na-K-2Cl cotransport (CT-kinase): both appear to be inhibited by cell swelling, NEM, and staurosporine.

The insensitivity of CT-kinase to norepinephrine and fluoride (Figs. 6 and 7) suggests that these stimuli, like calyculin A, activate Na-K-2Cl cotransport by inhibiting the deactivating phosphatase (PP). In support of this conclusion, we have found that dephosphorylation of the transporter after staurosporine inhibition of CT-kinase occurs much more slowly in cells stimulated by fluoride or norepinephrine than in cells stimulated by osmotic shrinkage. Fluoride is known to inhibit type 1 phosphatases in vitro at the concentrations employed in our experiments (4). Previous studies of duck red blood cells (31, 36) have provided good evidence that norepinephrine acts through cAMP via protein kinase A (PKA). One mechanism by which PKA could inhibit PP is by phosphorylating inhibitor-1, an endogenous protein inhibitor of type 1 protein phosphatases (4). Our results therefore suggest that the transporter is regulated in vivo through modulation of both the activating kinase (by cell volume) and the deactivating phosphatase (by PKA).

The nature of the volume-responsive kinase (its molecular identity, its own regulation, and its potential role in other osmosensitive processes) remains uncertain. These results demonstrate that CT-kinase is stimulated by cell shrinkage and suggest that it may also be stimulated by a product of its own reaction. We propose that CT-kinase activity might be enhanced by autophosphorylation. Finally, the results presented here indicate that it may be possible to isolate CT-kinase in an active and 32P-labeled form from shrunken duck red blood cells provided it is protected from dephosphorylation by calyculin A.

    ACKNOWLEDGEMENTS

I am grateful to Nichole McDaniel and Yeon Kim for expert technical assistance and to Dr. Bruce Cohen (University of California, Riverside, CA) for substantive suggestions on kinetic models. I thank Drs. Thomas J. McManus and Mark Haas for thoughtful discussions and critical review of the manuscript.

    FOOTNOTES

This work was supported by American Heart Association Grant-in-Aid AHA-94015270.

Received 22 October 1997; accepted in final form 12 December 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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