Na+-K+-2Clminus cotransport in Ehrlich cells: regulation by protein phosphatases and kinases

Thomas Krarup, Lene D. Jakobsen, Bo S. Jensen, and Else K. Hoffmann

Department of Biochemistry, The August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl- cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl- cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10% cell swelling and/or the concurrent increase in intracellular Cl- concentration. Cell shrinkage also activates the Na+-K+-2Cl- cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and Ca2+/calmodulin dependent. Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the Na+-K+-2Cl- cotransport activity during regulatory volume increase.

dephosphorylation; phosphorylation; bumetanide; myosin light chain kinase; protein kinase A; volume regulation

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ELECTRONEUTRAL Na+-K+-2Cl- cotransporter consists of at least two isoforms (reviewed in Refs. 7, 10, and 26). Na+-K+-2Cl- cotransport plays an important role in transepithelial transport of salt and water, and in symmetrical cells the cotransporter maintains cellular Cl- concentration above electrochemical equilibrium and restores cell volume after cell shrinkage (7, 10). Activation of Na+-K+-2Cl- cotransport is one of the first mitogenic signals (1), providing another important function of the cotransporter in symmetrical cells.

A variety of signal transduction pathways appear to be involved in regulation of Na+-K+-2Cl- cotransport (7, 10). Activation of cotransport by some agonists has been ascribed to the concomitant cell shrinkage or to the reduction in intracellular Cl- concentration ([Cl-]i) (10, 28), and shrinkage-induced activation of the Na+-K+-2Cl- cotransporter has been suggested to depend on [Cl-]i (10). Agonist-induced activation and cell shrinkage-induced activation of the cotransporter involve several protein kinases (27). The protein kinases involved in phosphorylation of the Na+-K+-2Cl- cotransport protein after cell shrinkage (8, 17, 24) remain unidentified.

Dephosphorylation of the Na+-K+-2Cl- cotransporter in avian erythrocytes is due to Ser/Thr protein phosphatase type 1 (PP-1) and/or type 2A (PP-2A), as estimated by the use of the protein phosphatase inhibitors okadaic acid (27) and calyculin A (Cal-A) (25). Cal-A equipotently inhibits PP-1, PP-2A, and PP-3 (11, 13). Cal-A activates Na+-K+-2Cl- cotransport, probably a consequence of increased net phosphorylation of the cotransporter (24), suggesting that Na+-K+-2Cl- cotransport is regulated by Ser/Thr kinases.

In aortic endothelial cells a 19-kDa component of the cytoskeleton, identified as myosin light chain (MLC), is phosphorylated in response to cell shrinkage (17); the phosphorylation is inhibited by the MLC kinase (MLCK) inhibitor ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1-hexahydro-1,4-diazepine], which at micromolar concentrations also inhibits shrinkage-activated Na+-K+-2Cl- cotransport. However, ML-7 is unable to block shrinkage-induced phosphorylation of the Na+-K+-2Cl- cotransporter in endothelial cells, suggesting that MLCK exerts its effect indirectly (17).

In Ehrlich cells the Na+-K+-2Cl- cotransporter mediates regulatory volume increase (RVI) (10) via a Ca2+/calmodulin (CaM)- and protein kinase C (PKC)-dependent mechanism (14). Inhibition of PKC, however, reduced cotransport activity only 20% during RVI (19), suggesting the involvement of other protein kinases.

We present pharmacological data suggesting continued protein phosphorylation/dephosphorylation of Ser/Thr residues in the Na+-K+-2Cl- cotransporter (or a regulatory protein) at steady state. We also show that MLCK and protein kinase A (PKA), directly or indirectly, participate in the isotonic steady-state phosphorylation of the Na+-K+-2Cl- cotransporter and that MLCK appears to modulate the Na+-K+-2Cl- cotransport activity during RVI.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells

Ehrlich ascites tumor cells (hyperdiploid strain) were maintained and harvested as described previously (14). The cytocrit was adjusted to 4 or 8%, and cells were kept at 37°C.

Incubation Media

Isotonic standard medium A was composed of (in mM) 150 Na+, 150 Cl-, 5 K+, 1 Ca2+, 1 Mg2+, 1 SO2-4, 1 HPO2-4, 3.3 MOPS, 3.3 TES, and 5 HEPES (~300 mosM), pH 7.4. Isotonic standard medium B was composed of (in mM) 150 Na+, 150 Cl-, 5 K+, 1 Ca2+, 1 Mg2+, 1 SO2-4, 1 HPO2-4, 5 N,N-bis(hydroxymethyl)glycine (bicene), and 5 N-tris(hydroxymethyl)methylglycine (tricene) (~300 mosM), pH 8.3. Hypertonic standard medium was composed of (in mM) 300 Na+, 300 Cl-, 10 K+, 2 Ca2+, 2 Mg2+, 2 SO2-4, 2 HPO2-4, 3.3 MOPS, 3.3 TES, and 5 HEPES (~600 mosM), pH 7.4. Strong hypertonic medium consisted of (in mM) 450 Na+, 450 Cl-, 15 K+, 3 Ca2+, 3 Mg2+, 3 SO2-4, 3 HPO2-4, 3.3 MOPS, 3.3 TES, and 5 HEPES (~900 mosM), pH 7.4. Buffered water consisted of (in mM) 3.3 MOPS, 3.3 TES, and 5 HEPES (~12 mosM), pH 7.4. All media were prewarmed to 37°C. When cells were preincubated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (35 min, final concentration 50 µM), the isotonic standard medium had a pH of 8.3 to circumvent the intracellular acidification caused by BAPTA-AM (15). In all other experiments (except Fig. 6) pH was 7.4.

Unidirectional K+ Influx

Na+-K+-2Cl- cotransport activity was measured as the bumetanide-sensitive, unidirectional K+ influx (in µmol · g dry wt-1 · min-1), as previously described (14), with 86Rb+ as a tracer for K+.

Before K+ influx measurements, cells (8% cytocrit) were preincubated in standard isotonic medium with one or a combination of protein phosphatase and kinase inhibitors before addition of experimental medium containing 86Rb+. To measure initial rates of K+ uptake in experiments examining the duration of the cotransport activity, 86Rb+ was added at different times after cellular stimulation (1, 5, 10, and 14 min). In the remaining experiments, 86Rb+ was present in the experimental medium.

Initial rates of K+ uptake were calculated by linear regression from four samples taken 0.5-3 min after addition of the isotope (14). Correlation coefficients between cellular counts per minute and time were >0.99.

Na+-K+-2Cl- cotransport activity during RVI is subject to considerable variation. We discarded results obtained from Ehrlich cells with a shrinkage-induced cotransport activity of <10 µmol · g dry wt-1 · min-1, since, because of the low stimulation of Na+-K+-2Cl- cotransport compared with isotonic controls (4 ± 0.6 µmol · g dry wt-1 · min-1, n = 18), the results on effects of protein kinase and phosphatase inhibitors would not be reliable.

Statistical significance was evaluated using Student's paired or independent t-test. Values are means ± SE.

Measurements of Cell Volume

Cell volume was measured using a Coulter counter (15).

Reagents

Reagents were of analytic grade; they were purchased from Sigma Chemical (St. Louis, MO) with these exceptions: 86RbCl was from Risø (Roskilde, Denmark), pimozide was a gift from Lundbeck (Copenhagen, Denmark), bumetanide was a gift from Leo Pharmaceuticals (Ballerup, Denmark), BAPTA-AM was from Molecular Probes (Eugene, OR), H-89, KT-5720, ML-7, ML-9, and staurosporine were from Calbiochem (Bad Soden, Germany), and KN-62, KN-04, Cal-A, deltamethrin, permethrin, and chelerythrine were from Alamone Labs (Jerusalem, Israel).

Stock solutions of chelerythrine (125 µM), 8-bromo-cAMP (5 mM), dibutyryl cAMP (100 mM), 8-bromo-cGMP (5 mM), and bradykinin (1 mM) were prepared in water. Pimozide (10 mM), bumetanide (10 mM), deltamethrin (100 µM), permethrin (500 µM), ML-7 (2 mM), and Cal-A (20 µM) were prepared in 96% ethanol. BAPTA-AM (10 mM), H-89 (1-2 mM), KN-62 (10 mM), KN-04 (10 mM), KT-5720 (200 µM), ML-9 (2 mM), and staurosporine (1-2 mM) were dissolved in desiccated DMSO. Stock solutions were stored at -20°C, except H-89, KN-04, KN-62, ML-7, and staurosporine, which were stored at 4°C. Cell suspensions received a maximum of 0.7% ethanol or 0.1% DMSO-0.3% ethanol (vol/vol). Controls received an appropriate volume of carrier(s).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Transient Activation of the Na+-K+-2Cl- Cotransporter

Na+-K+-2Cl- cotransport activity in unstimulated Ehrlich cells in isotonic medium was 4 ± 0.6 µmol · g dry wt-1 · min-1 (n = 18). Bradykinin (1 µM) stimulated cotransport fivefold (to 20 ± 1.5 µmol · g dry wt-1 · min-1, n = 11). In a strong hypertonic medium (final osmolarity 500 mosM) cotransport activity increased 7-fold (to 29 ± 2.0 µmol · g dry wt-1 · min-1, n = 5), and in a milder hypertonic medium (final osmolarity 400 mosM) the activity increased 5.5-fold (to 23 ± 2 µmol · g dry wt-1 · min-1, n = 22). This is accordance with previous estimations (14).

Maximal activation of the Na+-K+-2Cl- cotransporter appeared within the 1st min after addition of bradykinin, and the activity returned to the resting level ~5 min after stimulation (Fig. 1). Maximal activity of the Na+-K+-2Cl- cotransporter appeared rapidly after stimulation of the cells with a hypertonic medium with final osmolarity of 400 or 500 mosM (Fig. 1). In the 400 mosM medium the cotransport activity returned to the resting level within 8 min, whereas it declined more slowly toward the resting level in a strong hypertonic medium (500 mosM; Fig. 1), where it was still significantly higher than the basal level of activity (P <=  0.05, independent t-test) 10 min after the exposure. In contrast, cotransport activity 5 and 10 min after stimulation with bradykinin was not significantly different from the basal level (P >=  0.20, independent t-test; Fig. 1). Thus Na+-K+-2Cl- cotransport activity induced by strong hypertonic treatment outlasts the activity induced by bradykinin or by milder hypertonic challenge probably because of a longer-lasting cell shrinkage.


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Fig. 1.   Activity of Na+-K+-2Cl- cotransporter in Ehrlich ascites tumor cells after exposure to bradykinin or a hypertonic challenge. Na+-K+-2Cl- cotransport activity was measured as initial rates of K+ uptake 0.5-2 min after addition of 86Rb+ (50,000 Bq/ml) and is presented as unidirectional, bumetanide-sensitive K+ influx. When effect of bradykinin (1 µM) on cotransport activity was tested, 640-µl aliquots of cells (8% cytocrit) were preincubated with agent in isotonic medium. At time 0, cells were exposed to experimental medium including 30 µM bumetanide when appropriate. 86Rb+ was added after 1, 5, 10, and 14 min. Experimental conditions included exposure to hypertonic solutions of 400 (black-square) or 500 mosM (bullet ) or to bradykinin in isotonic medium (black-down-triangle ). Cotransport activity 1 min after a hypertonic challenge of 500 mosM was 19 ± 2.0 µmol · g dry wt-1 · min-1 (n = 3). Combined results from all experiments on bumetanide-sensitive K+ influx during regulatory volume increase and after inhibition with ML-7 and H-89 are included in Table 2. square , Cotransport activity in cells at physiological steady state during isotonic conditions. Values are means ± SE of 3-6 independent experiments. Shrinkage-induced activation of Na+-K+-2Cl- cotransporter was done using 2 hypertonic experimental media with final osmolarities of 400 and 500 mosM, respectively.

Effect of Protein Phosphatase Inhibitors on the Na+-K+-2Cl- Cotransporter Under Isotonic Conditions

The effect of PP-1, PP-2A, and PP-3 on Na+-K+-2Cl- cotransport activity was investigated using Cal-A. In in vitro experiments the inhibition constant (Ki) for Cal-A is in the nanomolar range for all three protein phosphatases (11, 13). The activity of the Na+-K+-2Cl- cotransporter increased with increasing concentrations of Cal-A (Fig. 2), and half-maximal activation (EC50) was obtained at ~35 nM. Maximal activation was obtained at >= 100 nM Cal-A.


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Fig. 2.   Dose-response relationship of calyculin A (Cal-A) activation of Na+-K+-2Cl- cotransporter. Ehrlich cells were preincubated in isotonic medium with Cal-A (0-400 nM) for 1 min. Values are means ± SE of 3 individual experiments.

Cal-A (100 nM) induced maximal activity of the Na+-K+-2Cl- cotransport activity within the 1st min (Fig. 3) at 43 ± 5 µmol · g dry wt-1 · min-1 (n = 3). In two independent experiments the bumetanide-sensitive K+ uptake was measured 15-60 s after addition of Cal-A (data not shown). The uptake was linear, indicating that maximal cotransport activity is achieved after 15 s of incubation with Cal-A. After 14 min of incubation with Cal-A the cotransport activity was not significantly different from the resting level (P >=  0.30, independent t-test; Fig. 3). Thus Cal-A-induced activation of the cotransporter is transient. This shows that some phosphatase activity remains after addition of Cal-A.


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Fig. 3.   Transient activation of Na+-K+-2Cl- cotransporter in Ehrlich cells exposed to Cal-A. Na+-K+-2Cl- cotransport activity was measured as described in Fig. 1 legend. Briefly, 86Rb+ was added 1, 5, 10, or 14 min after exposure of cells to Cal-A (100 nM); i.e., abscissa indicates duration of exposure to Cal-A. Uptake was measured 0.5-2 min after addition of 86Rb+, where uptake is linear (bullet ). square , Cotransport activity in unstimulated cells in isotonic medium. Values are means ± SE of 4-18 independent experiments.

To examine whether the inactivation of the Na+-K+-2Cl- cotransporter is secondary to cell swelling induced by Cal-A, cell volume was monitored using a Coulter counter. Cell volume increased as a result of the Cal-A-induced cotransport activity, promoting a net uptake of salt and water (Fig. 4). Inactivation of the Na+-K+-2Cl- cotransporter after Cal-A treatment, therefore, could be due to the increase in cell volume or, alternatively, to the concurrent increase in [Cl-]i. To investigate these possibilities, cells were preincubated in a hypotonic medium (225 mosM) for 5 min. One minute before addition of experimental medium containing 86Rb+, some of the cells also received 100 nM Cal-A, which stimulated the bumetanide-sensitive K+ influx from 11.1 to 21.5 µmol · g dry wt-1 · min-1 (n = 1). Thus Cal-A can activate the cotransporter even in cells swollen to ~1.1 times their normal volume. The activation was, however, smaller than when cells at normal volume are activated by Cal-A (Fig. 3).


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Fig. 4.   Effect of Cal-A on cell volume. Ehrlich cells (4% cytocrit) were diluted 1:200 in isotonic medium, and cell volume was measured using a Coulter counter. Cell volume is given as relative values (1 = 1,057 fl). Arrow, addition of 100 nM Cal-A (bullet ). Controls received only vehicle (open circle ). Results are representative of 3 individual experiments.

Whether inactivation of Na+-K+-2Cl- cotransport after prolonged exposure to Cal-A is due to cell lysis (32) was examined by light microscopy of cells exposed to Cal-A for 14 min. No changes in cell morphology were observed; i.e., cotransport inactivation 14 min after Cal-A exposure is not due to membrane deformation or cell lysis. The results with Cal-A suggest that PP-1, PP-2A, and/or PP-3 is involved in maintaining low Na+-K+-2Cl- cotransport activity under isotonic conditions at steady state.

Ca2+/CaM is involved in the regulation of the Na+-K+-2Cl- cotransporter (14). A possible role for the Ca2+/CaM-dependent PP-2B in the regulation of the cotransporter was therefore investigated using the potent PP-2B inhibitor deltamethrin (Ki = 30 pM in in vitro experiments) (5). The structurally related but inactive compound permethrin was used as a control. No significant difference in cotransport activity in cells preincubated with deltamethrin (100 nM, 15 min) or permethrin was observed (Table 1).The same concentration and preincubation time for deltamethrin were used in intact brain synaptosomes (5). Thus, although a positive control for the effect of deltamethrin in Ehrlich cells was lacking, a role for PP-2B in the inactivation of the cotransporter seems unlikely.

                              
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Table 1.   Effect of protein phosphatase inhibitors on Na+-K+-2Cl- cotransport activity

Effect of Protein Phosphatase Inhibitors on the Na+-K+-2Cl- Cotransporter After Stimulation With Bradykinin or Hypertonic Medium

Na+-K+-2Cl- cotransport activity after preincubation with deltamethrin or Cal-A and subsequent stimulation with bradykinin or a hypertonic challenge (500 mosM) was also evaluated. Deltamethrin had no effect compared with permethrin-treated cells and controls (Table 1). In contrast, bradykinin-induced cotransport activity was higher in Cal-A-treated cells than in cells exposed to bradykinin alone (P <=  0.01, independent t-test; Table 1). Similarly, cotransport activity during RVI was higher in Cal-A-treated cells than in untreated cells (P <=  0.05, independent t-test; Table 1).

Neither bradykinin nor hypertonicity could increase Na+-K+-2Cl- cotransport activity further in cells exposed to Cal-A (P >=  0.30, independent t-tests; Table 1). This indicates that Cal-A induces maximal activation of the cotransporter in Ehrlich cells.

Maximal activation of the cotransporter occurred within 1 min after stimulation with Cal-A plus bradykinin, with a decline toward the resting level during the next 10 min (Fig. 5A). After 5 min the cotransport activity was still significantly higher than the basal activity (P <=  0.05, independent t-test). Thus cotransport activity induced by Cal-A plus bradykinin was greater and lasted longer than that induced by bradykinin alone. In contrast, no significant difference was found between cells treated with Cal-A plus bradykinin and those treated with Cal-A alone. Therefore, the effects of Cal-A plus bradykinin on the Na+-K+-2Cl- cotransporter are not additive. Exposure to Cal-A, hypertonic medium, or Cal-A plus hypertonic medium activated cotransport maximally within 1 min, and the activity declined toward the resting level during the next 14 min (Fig. 5B). In cells exposed to Cal-A plus hypertonic medium, cotransport activity was significantly higher than at the resting level even after 14 min (P <=  0.05, independent t-tests), whereas neither cells treated with Cal-A nor those treated with hypertonic medium showed significantly increased activity after 10 min. Thus inactivation of cotransport is delayed when cells are exposed to Cal-A plus hypertonic medium compared with the inactivation in cells in hypertonic medium or after addition of Cal-A, indicating that PP-1, PP-2A, and/or PP-3 is involved in the inactivation of the Na+-K+-2Cl- cotransporter in agreement with the results in Table 1. However, other mechanisms must also be involved, since the process was delayed by Cal-A but not prevented. Alternatively, Cal-A may not inhibit the involved phosphatases completely.


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Fig. 5.   Effect of Cal-A on time course of bradykinin- or hypertonicity-activated Na+-K+-2Cl- cotransport activity. Na+-K+-2Cl- cotransport activity in Ehrlich cells was measured as described in Fig. 1 legend. Cal-A (100 nM) was added 1 min before bradykinin (1 µM) or hypertonic challenge (500 mosM), i.e., 1 min before time 0. 86Rb+ was added 1, 5, 10, and 14 min after exposure to bradykinin or hypertonic medium. square , Cotransport activity in unstimulated cells in isotonic medium. Values are means ± SE of 3-18 independent experiments. A: cotransport activity after stimulation with bradykinin (black-down-triangle ) or Cal-A (black-square). When cells were stimulated with Cal-A plus bradykinin (bullet ), Cal-A was added 1 min before bradykinin. B: cells exposed to Cal-A (black-square) or hypertonic medium (500 mosM, black-down-triangle ) and to Cal-A plus hypertonic medium (bullet ).

Role of Ca2+ and pH in Activation of the Na+-K+-2Cl- Cotransporter

The effect of chelating free intracellular Ca2+ with use of the cell-permeable Ca2+ chelator BAPTA-AM on the activation of the Na+-K+-2Cl- cotransporter was assessed. However, loading of Ehrlich cells with BAPTA provokes intracellular acidification. At extracellular pH (pHo) of 7.4, BAPTA resulted in an intracellular pH (pHi) of 6.5, whereas pHi in control cells was 7.2 (15). To compensate, BAPTA-loaded cells were incubated at pHo of 8.3, resulting in a pHi of ~7.2 (15). Cotransport activity after addition of bradykinin or during RVI was significantly lower in BAPTA-treated cells. Thus the initial activation of Na+-K+-2Cl- cotransport by bradykinin or hypertonicity depends on an increase in intracellular Ca2+ concentration ([Ca2+]i) or on a certain basal [Ca2+]i. The basal [Ca2+]i is decreased from 59 ± 2 nM (controls) to 26 ± 1 nM in BAPTA-loaded cells (15).

Preliminary experiments at pHo of 7.4 showed that BAPTA-induced acidification caused a marked decrease in cotransport activity. The pH dependence of the cotransport activity was thus further investigated. At pHo of 6.5, cotransport activity during RVI was negligible but increased with increasing pHo to 24 ± 3.9 µmol · g dry wt-1 · min-1 at pHo of 8.3 (Fig. 6). The decrease in cotransport activity caused by BAPTA-induced acidification suggests that the inhibitory effect of H+ is internal. The data in Fig. 6 exclude that the inhibitory effect of BAPTA in Table 2 is due to the exposure of the cells to a pHo of 8.3. 


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Fig. 6.   Effect of pH on Na+-K+-2Cl- cotransport activity after a hypertonic challenge. Ehrlich cells were preincubated for 10 min in isotonic media at pH 6.5, 6.9, 7.4, and 8.3. Na+-K+-2Cl- cotransport activity was measured after a hypertonic challenge (400 mosM) with experimental media (containing 86Rb+) at pH values identical to those of incubation media. Values are means ± SE of 3 separate experiments.

                              
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Table 2.   Effect of protein kinase inhibitors on Na+-K+-2Cl- cotransport activity

Ca2+/CaM and PKC Are Involved in Activation of the Na+-K+-2Cl- Cotransporter

Previous reports have suggested that in Ehrlich cells the Ca+/CaM complex and PKC are involved in cotransport activation during RVI (14, 19).

Bradykinin-induced cotransport activity was inhibited by 56% in cells pretreated with 100 µM pimozide (P <=  0.0, paired t-test), slightly, but not significantly, inhibited (P >=  0.40, independent t-test) after preincubation with chelerythrine (2.5 µM), and inhibited by 75% when the two inhibitors were combined (Table 2). In the presence of both inhibitors the cotransport activity was significantly lower than after separate preincubation with pimozide or chelerythrine (P <=  0.0, paired t-tests) and not significantly different from the activity in unstimulated cells. This pharmacological evidence indicates the involvement of a Ca2+/CaM and PKC in the cotransport activation.

CaM Kinase II Is Not Involved in Activation of the Na+-K+-2Cl- Cotransporter

Ca2+/CaM may influence the Na+-K+-2Cl- cotransporter through the multifunctional Ca2+/CaM-dependent kinase CaM kinase II. No inhibitory effect of the specific CaM kinase II inhibitor KN-62 (10 µM) on cotransport activity could, however, be demonstrated (Table 2). KN-04, a structurally related but inactive analog, was used as a control. Therefore, CaM kinase II is apparently not involved in the regulation of the cotransporter in Ehrlich cells. However, no positive control for the effect of KN-62 on CaM kinase II in Ehrlich cells has been conducted; thus the results are not conclusive. KN-62 was, however, used in a concentration 10 times higher than the Ki for CaM kinase II in vitro (31) and than the effective concentration used on PC-12D cells (31) and thus should be effective also on Ehrlich cells.

MLCK and PKA Are Involved in Activation of Na+-K+-2Cl- Cotransport After Hypertonic Stimulation

Figure 7 shows the effect of 1 µM ML-7 on the time course of the unidirectional, bumetanide-sensitive K+ uptake in Ehrlich cells during RVI in a hypertonic medium (400 mosM). ML-7 specifically inhibits the Ca2+/CaM-dependent Ser/Thr kinase MLCK, with a Ki of 0.3 µM (30). Bumetanide-sensitive K+ uptake in cells at isotonic steady state was included as a control. The increase in the rate of bumetanide-sensitive K+ uptake in hypertonic medium was significantly inhibited (71%) by ML-7 (Fig. 7). The extent of inhibition may be slightly overestimated, since any possible effect of ML-7 on the basal K+ uptake was neglected. Table 2 summarizes the results of nine independent experiments in the presence of ML-7 (1 µM). In six paired experiments, ML-7 inhibited cotransport activity during RVI by 50 ± 3% (n = 6, P = 0.003, paired t-test) or the cotransport activity increase in hypertonic medium by 66%, suggesting that MLCK is likely to participate in regulation of the cotransporter during RVI. H-89 inhibits PKA with a Ki of 48 nM (2), and staurosporine is a nonspecific inhibitor of many protein kinases, including Ser/Thr protein kinases (29). In four paired experiments, H-89 (1 µM) inhibited cotransport by 43 ± 7% (P = 0.007, paired t-test), indicating a possible role for PKA in the regulation of Na+-K+-2Cl- cotransport during RVI. Pretreatment of the cells with ML-7 plus H-89 in three paired experiments resulted in a nearly threefold decrease in cotransport activity to 36 ± 4% (P = 0.011, paired t-test; Table 2). A similar cotransport inhibition was observed in cells exposed to 1 µM staurosporine (to 34 ± 6%, n = 6, P = 0.001, paired t-test; Table 2). The residual, shrinkage-activated K+ influx in the presence of staurosporine, approximately one-third of the total, appears to be controlled by pathways not involving Ser/Thr kinases.


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Fig. 7.   Effect of ML-7 on Na+-K+-2Cl- cotransport activity in Ehrlich cells in hypertonic media. Time course of K+ uptake via Na+-K+-2Cl- cotransporter in isotonic (down-triangle) or hypertonic medium (400 mosM): cells were preincubated for 3 min with ML-7 (bullet ) or carrier (open circle ). Unidirectional K+ uptake over 1-3 min is essentially linear, indicating that samples taken within this interval provide good data for calculation of influx rates. Similar results were obtained in 7 experiments of the same design, which gave a bumetanide-sensitive K+ influx of 13.8 ± 1.8 (n = 8) and 2.2 ± 0.5 µmol · g dry wt-1 · min-1 (n = 8) during regulatory volume increase (RVI; at 400 mosM) and at steady state (at 300 mosM), respectively. In presence of ML-7 during RVI, a bumetanide-sensitive K+ influx of 10 ± 1.2 µmol · g dry wt-1 · min-1 (n = 6) was obtained. Only 2 experiments included all 3 experimental conditions tested, and 1 experiment testing effect of ML-7 on RVI-activated Na+-K+-2Cl- cotransport was discarded, because bumetanide-sensitive K+ influx was <= 10 µmol · g dry wt-1 · min-1 compared with mean isotonic controls of 4 ± 0.6 µmol · g dry wt-1 · min-1 (n = 18; Table 1).

Because neither ML-7 nor H-89 is entirely specific for MLCK and PKA, respectively (Calbiochem specifications), dose-response experiments were performed for all four inhibitors (Fig. 8). The results support those obtained using ML-7 and H-89. The observed IC50 values are summarized in Table 3.


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Fig. 8.   Dose-response curves for inhibitors of myosin light chain kinase (MLCK) and protein kinase A (PKA) of Na+-K+-2Cl- cotransport activity in Ehrlich cells in hypertonic media. Cells were preincubated for 3 min in isotonic standard medium with 0-36 µM ML-7 (A) and ML-9 (B), MLCK inhibitors, or with 0-8 µM H-89 (C) and KT-5720 (D), PKA inhibitors. At time 0 a hypertonic experimental medium (final osmolarity 400 mosM) containing 86Rb+ and bumetanide, when appropriate, was added. Na+-K+-2Cl- cotransport activity was measured as bumetanide-sensitive unidirectional K+ influx. A-D show result of 1 dose-response experiment (open circle ) in which 1 protein kinase inhibitor was used. To calculate IC50, each set of experimental data was fitted using nonlinear least-squares iterative procedures (SigmaPlot, Jandel, Corte Madera, CA) to the following equation: J = Jmax - {(Jmax - Jmin)/(1 + IC50/[I])}, where J is measured bumetanide-sensitive K+ influx, Jmax is measured bumetanide-sensitive K+ influx in absence of protein kinase inhibitor, Jmin is protein kinase inhibitor-insensitive K+ influx, and [I] is protein kinase inhibitor concentration. Fits (solid lines) gave estimates of Jmin and IC50 ± asymptotic SE. Each experiment was repeated, and experimental data were fit individually to equation. Values (means ± SE) for Jmax and Jmin (in µmol K+ · g dry wt-1 · min-1) were 732.5 ± 3.5 and 18.9 ± 1.6, respectively, for ML-7 (n = 3), 28.2 ± 1.3 and 9.2 ± 1.7, respectively, for ML-9 (n = 3), 27.7 ± 7.0 and 14.3 ± 3.3, respectively, for H-89 (n = 2), and 30.5 ± 5.5 and 12.0 ± 2.6, respectively, for KT-5720 (n = 3). Means of IC50 values are summarized in Table 3.

                              
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Table 3.   IC50 for MLCK and PKA inhibitors on Na+-K+-2Cl- cotransport activity in Ehrlich cells in hypertonic media

Because inhibition of PKA by H-89 decreases Na+-K+-2Cl- cotransport activity during RVI (Table 2), the effect of stimulation of PKA with forskolin (300 µM) was tested on the cotransport activity during RVI. Forskolin had no effect, indicating that cAMP may not be rate limiting (Table 4).

                              
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Table 4.   Effect of cyclic nucleotides on Na+-K+-2Cl- cotransport activity

Cal-A-Activated Na+-K+-2Cl- Cotransport Is Regulated by MLCK, PKA, and Other Ser/Thr Kinases Under Isotonic Conditions

The Na+-K+-2Cl- cotransporter at isotonic steady state is regulated by continuous phosphorylation and dephosphorylation; our pharmacological evidence suggests that dephosphorylation is due to PP-1, PP-2A, and/or PP-3 (Table 1). Under isotonic steady-state conditions, cotransport activity is very low (Table 1), making it difficult to investigate the effect of protein kinase inhibitors. Therefore, the possible role of MLCK, PKA, and other Ser/Thr kinases in the steady-state phosphorylation of the cotransporter was measured in Cal-A-treated cells. The Na+-K+-2Cl- cotransport activity obtained after 1 min of preincubation with Cal-A is regarded as a steady-state value, since maximal Cal-A-induced cotransport was obtained within 15 s and was constant in 15 s-1 min (see above). Because the cotransport activation is transient (Fig. 3), the decrease in cotransport activity is regarded as a result of secondary inhibition of a kinase caused by cell swelling (Fig. 4) and/or an increase in [Cl-]i. In the presence of a kinase inhibitor that partially inhibits the activating kinase, we expected maximal cotransport activation to be reached, but at a slower rate. Thus after preincubation for 3 min with a kinase inhibitor and 1 min with Cal-A, a new steady-state situation is expected, where the lower activity of the cotransporter reflects a lower net phosphorylation. It therefore seemed reasonable to test which kinases are involved in the steady-state cotransport activity after 1 min of preincubation with Cal-A with use of the available pharmacological tools with the limitations of this approach.

Cells were preincubated with Cal-A (control) or Cal-A plus one or two Ser/Thr protein kinase inhibitors. Relative to the controls (no protein kinase inhibitor), K+ influx via the Na+-K+-2Cl- cotransporter in four paired experiments was reduced to 0.71 ± 0.02 (P <=  0.001) after addition of ML-7, 0.76 ± 0.05 (P <=  0.006, paired t-test) after addition of H-89, and 0.65 ± 0.03 (n = 3, P <=  0.009, paired t-test) after addition of ML-7 plus H-89. In five other paired experiments, staurosporine reduced Cal-A-activated Na+-K+-2Cl- cotransport activity to 0.50 ± 0.05 (P <=  0.002, paired t-test; for absolute values see Table 2). Inhibition of PP-1, PP-2A, and/or PP-3 by Cal-A thus results in a net phosphorylation caused by MLCK, PKA, and other Ser/Thr kinases during RVI and at steady state.

MLCK-Sensitive Cotransport Increases After Cell Shrinkage

Figure 9 shows the ML-7-sensitive part of Na+-K+-2Cl- cotransport activity in cells during RVI or pretreated with Cal-A in isotonic standard medium. The results, calculated as the difference in bumetanide-sensitive K+ influxes in the presence or absence of protein kinase inhibitor on the same batch of cells, show that the ML-7-sensitive cotransport activity is almost twofold larger during RVI than after Cal-A treatment: 6 ± 0.4 (n = 4) vs. 12 ± 1.7 µmol · g dry wt-1 · min-1 (n = 9, P = 0.069, independent t-test). This indicates that cell shrinkage activates an ML-7-sensitive Na+-K+-2Cl- cotransport. In contrast, the H-89-sensitive part of the K+ influx remained unchanged during RVI whether it was tested in four paired experiments (P > 0.4, paired t-test) or as an unpaired t-test (P = 0.2, n = 4 for Cal-A-treated cells and n = 6 during RVI). Our results with forskolin indicate that neither activation of PKA during isotonic steady state nor activation of PKA during RVI increases cotransport activity further (Table 4). The larger ML-7-sensitive K+ influx during RVI suggests that the enhanced cotransport activity is due not only to the protein kinases (including MLCK and PKA) that maintain the steady-state phosphorylation but also to an additional activation of MLCK (Fig. 9), thus emphasizing the possible role of MLCK as a modulator of the Na+-K+-2Cl- cotransporter under hypertonic conditions.


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Fig. 9.   Effect of ML-7 on Cal-A- or shrinkage-induced Na+-K+-2Cl- cotransport activity. Data are presented as difference in bumetanide-sensitive K+ influxes between measurements with and without ML-7 (1 µM) in the same cell batch, i.e, inhibitor-sensitive Na+-K+-2Cl- cotransport activity. Mean estimates for ML-7-sensitive part of cotransport activity were 6 ± 0.4 (n = 4) and 11.5 ± 1.7 µmol · g dry wt-1 · min-1 (n = 9) after Cal-A treatment (hatched bar) and during RVI (open bar), respectively. ML-7-sensitive cotransport activity is 2 times higher after cell shrinkage than after Cal-A treatment, although result is not quite significant at 0.05 level (P = 0.069, independent t-test).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Activation of the Na+-K+-2Cl- Cotransporter in Ehrlich Cells

The Na+-K+-2Cl- cotransporter in Ehrlich cells is stimulated by bradykinin or by exposure of cells to a hypertonic challenge. Activation of cotransport after cell shrinkage in hypertonic medium and activation after addition of bradykinin have been reported in many cell types (10, 14, 23). In Ehrlich cells, bradykinin induces a biphasic cell shrinkage. Original cell volume is restored within 5 min (10). Bradykinin-induced cotransport activity in Ehrlich cells reaches maximal activity shortly after stimulation and returns to the basal level within ~5 min (Fig. 1). Prevention of thrombin- or bradykinin-induced cell shrinkage by charybdotoxin abolished activation (10). Thus the effect of bradykinin on cotransport may be due to bradykinin-induced cell shrinkage.

In cells exposed to hypertonic treatment the cotransport activity rapidly increases to a maximal level and then declines to the basal level after ~15 min in strong hypertonic medium (500 mosM) or after ~9 min in a milder hypertonic medium (400 mosM; Fig. 1). This finding suggests that the duration of the increased cotransport activity after hypertonic treatment depends on the osmolarity and, hence, the degree of cell shrinkage. Na+-K+-2Cl- cotransport activity induced by the 500 mosM hypertonic medium also lasts longer than the cotransport activity induced by bradykinin, reflecting that the cells are shrunken for a longer period after addition of hypertonic medium than after addition of bradykinin.

Pharmacological Evidence for Involvement of Different Protein Phosphatases in Regulation of the Na+-K+-2Cl- Cotransporter Under Isotonic Conditions

PP-1, PP2A, and/or PP-3 appears to be involved in dephosphorylation of the Na+-K+-2Cl- cotransporter in Ehrlich cells under steady-state conditions. Inhibition of these phosphatases by Cal-A causes a pronounced increase in cotransport activity under isotonic conditions (Figs. 2 and 3), indicating that these protein phosphatases are constantly active. Stimulation of Na+-K+-2Cl- cotransport activity by Cal-A has been reported in other cell types, e.g., vascular endothelial cells (18, 24), PC-12 cells (20), avian erythrocytes (25), and brain capillary endothelial cells (32). Na+-K+-2Cl- cotransport activity increases with increasing Cal-A concentration (Fig. 2). Maximal activity is obtained at >= 100 nM Cal-A. EC50 is close to 35 nM, which is comparable to EC50 values found in other cell types (20, 25, 32). The effect of Cal-A is rapid (Fig. 3), as shown in avian erythrocytes (25). Activation of the Na+-K+-2Cl- cotransporter by Cal-A is biphasic and lasts <14 min in Ehrlich cells (Fig. 3), in contrast to observations on avian erythrocytes, where the cotransport activity remains high for at least 60 min after addition of Cal-A (25). Inactivation of the Na+-K+-2Cl- cotransporter after Cal-A exposure could reflect the observed 10% increase in cell volume (Fig. 4) and/or the concurrent increase in [Cl-]i. In PC-12 cells, cell volume also increases immediately after addition of Cal-A (20), as demonstrated here for Ehrlich cells (Fig. 4). The following results argue against the 10% increase in cell volume as a "turn-off signal" for the cotransporter. 1) During regulatory volume decrease the cotransporter is slightly stimulated after 5 min, even though cell volume is still ~1.1 times the normal volume (14). 2) Cal-A also stimulates the Na+-K+-2Cl- cotransporter in cells swollen in hypotonic media to 1.1 times their normal volume (n = 1, result not shown). Thus the increase in [Cl-]i after addition of Cal-A to cells in isotonic medium may be the turn-off signal for the Na+-K+-2Cl- cotransporter. [Cl-]i as a regulator of the Na+-K+-2Cl- cotransporter in Ehrlich cells has previously been suggested (see Ref. 10 for references). Recent evidence demonstrating increased phosphorylation of the cotransport protein itself as a consequence of low [Cl-]i strongly supports this hypothesis (8). [Cl-]i will increase with cell swelling resulting from an uptake of KCl. The cotransport activation by Cal-A in swollen cells is, however, still small compared with the activation in cells at normal cell volume; i.e., the change in cell volume also has a strong modulating effect on the cotransport activity.

Deltamethrin had no effect on the Na+-K+-2Cl- cotransport activity (Table 1). Provided that deltamethrin has access to PP-2B in Ehrlich cells (see RESULTS), we propose that PP-2B is not involved in regulation of Na+-K+-2Cl- cotransport. To our knowledge, a role for PP-2B in the regulation of the Na+-K+-2Cl- cotransporter has not been investigated in other cell types.

Involvement of Different Protein Phosphatases in Regulation of the Na+-K+-2Cl- Cotransporter After Stimulation With Bradykinin or Addition of Hypertonic Medium

The combined effect of bradykinin and Cal-A on Na+-K+-2Cl- cotransport activity is larger and longer lasting than the effect of bradykinin alone (Fig. 5A). The same scenario is observed when the effect of hypertonic medium plus Cal-A is compared with the effect of hypertonic medium or Cal-A alone (Fig. 5B), consistent with the hypothesis that the inactivation seen after stimulation with Cal-A is partly a consequence of cell swelling and that PP-1, PP-2A, and/or PP-3 seems to be involved in the inactivation of the cotransporter.

Involvement of Different Protein Kinases in Regulation of the Na+-K+-2Cl- Cotransporter Under Isotonic Conditions

The Na+-K+-2Cl- cotransporter is an example of a protein that is regulated by reversible phosphorylation. The cotransporter is quiescent under isotonic steady-state conditions, with the steady-state level of phosphorylation maintained by regulation of the activities of protein kinase(s) and phosphatase(s). Activation of the Na+-K+-2Cl- cotransport by Cal-A treatment in Ehrlich cells and subsequent reduction by protein kinase inhibitors confirm the results of others (18, 20, 25, 32). In Ehrlich cells, steady-state phosphorylation of the cotransporter is apparently dependent on MLCK and PKA (Table 2) and the dephosphorylation process on the Cal-A-inhibitable PP-1, PP-2A, and/or PP-3 (Fig. 2). In avian erythrocytes, PKA and cAMP-independent kinases possibly are maintaining steady-state phosphorylation of the Na+-K+-2Cl- cotransporter (27).

Involvement of Different Protein Kinases in Activation of the Na+-K+-2Cl- Cotransporter During RVI and After Agonist Stimulation

Ca2+/CaM-dependent mechanisms. Previously we found that activation of Na+-K+-2Cl- cotransport during RVI is Ca2+/CaM dependent (14). Here we demonstrate that cotransport activity induced by bradykinin or cell shrinkage is markedly reduced in cells loaded with BAPTA, an intracellular chelator of Ca2+ (Table 2). BAPTA loading of Ehrlich cells decreases intracellular Ca2+ concentration ([Ca2+]i) by ~50% compared with control cells but also causes an immediate intracellular acidification (15), which itself inhibits cotransport (Fig. 6). Preincubation of cells in a medium of pH 8.3 prevents the intracellular acidification (15) without preventing inhibition of the cotransport activity by BAPTA. Cell shrinkage due to a loss of K+ and Cl- presumably via the otherwise silent K+-Cl- cotransporter is another effect of BAPTA loading (15). However, it is unlikely that this cell shrinkage results in an inhibition of the Na+-K+-2Cl- cotransporter. Therefore, activation of Na+-K+-2Cl- cotransport depends on [Ca2+]i, as indicated by the absence of cotransport in BAPTA-loaded cells (Table 2). A substantial decrease in agonist-induced cotransport after preincubation with BAPTA-AM is also found in endothelial cells (23) and nasal gland acinar cells (12). In Ehrlich cells no increase in [Ca2+]i during RVI could be demonstrated (14), although minor spatially localized increases in [Ca2+]i cannot be excluded by the method used.

The Na+-K+-2Cl- cotransport activity in Ehrlich cells is abolished at pHo of 6.5 but increases with increasing pHo. Hence, the cotransport is dependent on pH, probably pHi (Fig. 6; see RESULTS). In mouse kidney it was shown that the cotransporter of the medullary thick limb of Henle is inhibited by intracellular H+ concentration (16), similar to the results obtained here. In duck erythrocytes, bumetanide binding and cotransport activity are dependent on pH (9).

The Ca2+/CaM antagonist pimozide inhibits bradykinin-stimulated Na+-K+-2Cl- cotransport activity by 71% (Table 2). Although nonspecific effects of pimozide cannot be excluded, a role for CaM in activation of the cotransport is indicated. The result is comparable to the previously reported 73% inhibition by pimozide of cotransport activity during RVI in Ehrlich cells (14). Accordingly, agonist stimulation did not activate Na+-K+-2Cl- cotransport in endothelial cells preincubated with W-7, another CaM antagonist (23). No effect of the CaM kinase II inhibitor KN-62 could be demonstrated, supporting the results of Ikeda et al. (12).

PKC. In Ehrlich cells, PKC is maximally activated 1 min after exposure to a hypertonic medium, and chelerythrine, a specific PKC inhibitor, inhibits cotransport by 20% during RVI (19). In contrast, no significant decrease in bradykinin-induced cotransport activity could be demonstrated here (Table 2). However, the bradykinin-induced Na+-K+-2Cl- cotransport activity is significantly lower in cells treated with pimozide plus chelerythrine than in cells treated with pimozide alone (Table 2). This suggests that PKC plays a minor role in activation of the Na+-K+-2Cl- cotransporter in Ehrlich cells. In general, the role of PKC in the regulation of the cotransporter is not clear, since, depending on cell type, activation of PKC can be stimulatory or inhibitory (10).

MLCK. It has been suggested that MLCK is important for Na+-K+-2Cl- cotransport during RVI in endothelial cells (17, 24): the cotransport activity on cell shrinkage was inhibited ~30 and 50% at 25 and 100 µM ML-7, respectively, but was not inhibited at 1 µM ML-7. At 100 µM, ML-7 blocks MLC phosphorylation and Na+-K+-2Cl- cotransport activity, but not phosphorylation of the cotransporter itself (17). However, according to Calbiochem specifications, ML-7 also inhibits PKA and PKC, with Ki values of 21 and 42 µM, respectively; thus it is difficult to make definite conclusions as to which kinase is involved in regulation of cotransport activation at 100 µM ML-7.

In Ehrlich cells, ML-7 and ML-9 decreased Na+-K+-2Cl- cotransport activity during RVI, with IC50 values of 0.4 and 2.8 µM, respectively, for the inhibitor-sensitive part of the influx (Table 3), values close to the Ki values reported for ML-7 and ML-9 inhibition of MLCK: 0.3 and 3.8 µM (30). The pharmacological evidence thus suggests that MLCK is a major modulator of the shrinkage-induced activation of the cotransporter in Ehrlich cells.

PKA. PKA inhibitors H-89 and KT-5720 reduced Na+-K+-2Cl- cotransport during RVI, with IC50 values of 37 and 34 nM, respectively, for the inhibitor-sensitive part of the influx (Table 3). The IC50 values are close to the reported Ki values at 48 and 60 nM for H-89 (2) and KT-5720 (6), respectively. On the basis of these pharmacological data, we propose that PKA is involved in the regulation of the cotransporter in Ehrlich cells. Interestingly, activation of PKA during isotonic steady state or RVI does not result in enhanced cotransport activity (Table 4). In duck erythrocytes, PKA has likewise been assigned a role as modulator of Na+-K+-2Cl- cotransport on the basis of effects of the inhibitors H-9 [N-(2-aminoethyl)-5-isoquinolinesulfonamide] and K-252a (27). The role of cAMP and, therefore, PKA is ambiguous: cAMP stimulates cotransport in some avian cells, whereas in other cell types, such as fibroblasts, cotransport is inhibited by cAMP (7, 10). PKA is not likely to be the protein kinase controlling the RVI response (27). As shown in the present report, the H-89-sensitive part of the cotransport activity does not increase significantly after cell shrinkage, suggesting that PKA is involved in the maintenance of steady-state phosphorylation without being further activated during RVI.

Activation of MLCK on Cell Shrinkage

In contrast to the H-89-sensitive part of cotransport, the ML-7-sensitive part increases nearly twofold during RVI compared with the activity at steady state (Fig. 9). This suggests that MLCK is further activated during RVI; i.e., cell shrinkage seems to activate MLCK.

MLCK is a Ca2+/CaM-dependent kinase; hence, the fact that pimozide and BAPTA strongly inhibit cotransport activity during RVI (Table 2) supports the suggested role for MLCK. Thus the present pharmacological data suggest that the regulatory pathway involved in shrinkage activation of the cotransporter involves activation of a volume-sensitive Ca2+/CaM-dependent MLCK and concomitant phosphorylation of MLC. MLC phosphorylation, however, may not be due to MLCK only: PKC is also active during RVI (19) and has MLC among its substrates. The PKC inhibitor chelerythrine and the Ca2+/CaM antagonist pimozide seem to have additive effects (Table 2), suggesting that the activity of PKC is unrelated to MLCK. Whether the effects of ML-7 and chelerythrine are additive has not been investigated. Further investigations, based on molecular and biochemical evidence, are needed to characterize the role of MLCK in the regulation of the Na+-K+-2Cl- cotransporter.

How Is MLCK Activated?

Two major theories for explaining how cells sense volume alterations and activate ion transport systems have been put forward. The first theory involves the macromolecular crowding of cytosolic solutes (10): on the basis of the inhibitory effect of urea, Lim et al. (21) suggested that Na+-K+-2Cl- cotransport in human erythrocytes may indeed be regulated by macromolecular crowding. The second theory suggests that the cytoskeletal structure is involved in the signal transduction (22). Phosphorylation affects cytoskeletal assembly, and MLCK is tightly associated with cytoskeletal proteins (4). There is evidence that exposure of Ehrlich cells to cytochalasin B (which causes actin filament disassembly) before cell shrinkage decreases Na+-K+-2Cl- cotransport activation during RVI by ~41% compared with controls (10). This is similar to the inhibitory effect obtained here with ML-7 of 47% (Fig. 8A), suggesting that the influence of MLCK on Na+-K+-2Cl- cotransport activity during RVI may be exerted via the cytoskeleton. In sheep erythrocytes the structurally related K+-Cl- cotransporter is activated by two different signals on cell swelling: one is sensed as a reduction in macromolecular crowding and the other is mechanical (3). The latter signal, therefore, could include reorganization within the cytoskeleton.

In conclusion, using various pharmacological tools, we have demonstrated that the protein kinases MLCK and PKA participate in the continuous phosphorylation and the protein phosphatases PP-1, PP-2A, and/or PP-3 in the dephosphorylation of the Na+-K+-2Cl- cotransporter (or a regulatory protein) during isotonic steady state in Ehrlich cells. In addition, we found that the ML-7-sensitive cotransport is further activated after cell shrinkage, indicating that an ML-7-sensitive process, most probably MLCK, plays a regulatory role during RVI in Ehrlich cells.

    ACKNOWLEDGEMENTS

The expert technical assistance of Birgit B. Jørgensen is acknowledged.

    FOOTNOTES

This research was supported by Carlsberg Foundation Grants 960305/20-1299 and 960344/40-1099, The Novo Foundation, and Danish Natural Science Research Council Grants 110702-2 and 111228-1.

Address reprint requests to E. K. Hoffmann.

Received 28 October 1996; accepted in final form 30 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 275(1):C239-C250
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