Na+ and K+ regulate the phosphorylation state of nucleoside diphosphate kinase in human airway epithelium

L. J. Marshall, R. Muimo, C. E. Riemen, and A. Mehta

Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We describe how cations, in the presence of ATP, regulate the phosphorylated form of 19- and 21-kDa nucleoside diphosphate kinase (NDPK; EC 2.7.4.6), a kinase controlling K+ channels, G proteins, cell secretion, cellular energy production, and UTP synthesis. In apically enriched human nasal epithelial membranes, 10 mM Na+ inhibits phosphorylation of NDPK relative to other cations. Dose response showed that, whereas K+ induces a fourfold greater phosphate incorporation (EC50 10 mM), Na+ is inhibitory (EC50 10 mM) compared with respective buffer controls. Cation discrimination is nucleotide selective (not seen with [gamma -32P]GTP) and NDPK specific (not seen with p37h, a previously characterized Cl--sensitive phosphoprotein). Na+ does not exert an inhibitory effect on NDPK phosphorylation directly but is likely to act via an okadaic acid-insensitive phosphatase. We speculate that the ability of NDPK to discriminate between physiologically relevant cation concentrations provides a novel example of cross talk within the apical membrane.

membrane; phosphatase; nucleotide; adenosine 5'-triphosphate; chloride

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE REGULATION OF Na+ absorption from the apical surface of epithelial cells involves the coordination of the following processes: Na+ entry to the subapical cytoplasm via Na+ channels, consumption of energy for maintenance of a steady intracellular Na+ concentration ([Na+]i) via activation of the basolaterally located Na+-K+-ATPase, and prevention of the rise in intracellular K+ concentration ([K+]i) by extrusion through basolateral K+ channels (17, 32). Neither the process (which involves cross talk) nor the molecular linkages are understood. Here we describe the properties of a prerequisite for a coherent model: a kinase differentially regulated by [Na+]i and [K+]i.

There are at least three (nonexclusive) ways in which Na+ may control its own transepithelial flux. These include 1) inhibition of Na+ entry ("self-inhibition") by extracellular Na+ concentration ([Na+]o; see review in Ref. 32), 2) regulation of Na+ channel recruitment from subapical pools (34), and 3) [Na+]i acting on an intracellular signaling pathway, a notion we expand in this paper. Thus Komwatana et al. (15) described saturation of Na+ conductance as the [Na+]o increased in mouse mandibular duct cells, and Turnheim (32) has reviewed the evidence for an external Na+ sensor. Van Driessche and Lindemann (34) found that [Na+]o-dependent saturation of Na+ transport was due to a decline in Na+ channel density, a process that Els and Chou (7) found to be sensitive to cytochalasin B in frog skin. However, the simple interpretation that the density of Na+ channels, and therefore the rate of Na+ transport, relied on (microfilament-dependent) recruitment of new channel proteins has been challenged by recent studies (1). The new data suggest that cytochalasin B may inhibit the translocation of an inhibitory protein kinase to the channel. The authors of these studies showed (using Xenopus oocytes and planar lipid bilayers) that activity of the amiloride-blockable epithelial Na+ channel was inhibited by protein kinase C (PKC) but found that inhibition was 80% attenuated by pretreatment with cytochalasin B. These experiments introduced the idea that PKC-dependent phosphorylation is involved in the inhibition of Na+ channel activity, but it should be noted that, in this system, protein kinase A did not activate the channel. The third model speculates that Na+ interacts with second messengers. This model predicts an intracellular sensor(s) for Na+ within a signaling cascade(s), an idea we explore here by defining the relationship between the degree of phosphorylation of a kinase [nucleoside diphosphate kinase (NDPK)] and the cation species bathing apical membrane fractions derived from the Na+-absorptive epithelium of the human upper airway.

The current study complements our earlier demonstration of a phosphorylation-mediated, Cl- concentration ([Cl-])-dependent sensor cascade in human (30, 31) and sheep airway epithelium (21). We reported that the rate of change in the in vitro phosphorylation profile of a 37-kDa protein (p37h or p37s; suffixes denote human and sheep, respectively) was maximal on either side of the steady-state [Cl-]i in airway epithelial cells (~40 mM; see Ref. 30). We speculated that the role of the membrane-bound kinase(s) and or phosphatase(s) regulating the net phosphorylation of p37h or p37s could be to "feed back" [Cl-]i to membrane transporters and/or channels, which would explain the well-established connection between [Cl-]i and the membrane conductance of Na+ and Cl- (5, 6, 18, 20, 29, 36). During the course of these studies, we found that Na+, in a concentration range expected for the epithelial cytoplasm (~10 mM), inhibited apical membrane phosphorylation (Mehta, unpublished observations). The present study had two aims, 1) to study the effects of cations on apical membrane phosphorylation by clamping [Cl-] below the concentration that affects phosphorylation and 2) to show that Cl- interacts with Na+ and K+ to exert, respectively, inhibitory and permissive effects on NDPK phosphorylation. We observe that NDPK (but not p37h) is able to discriminate between K+ and Na+ at physiologically relevant intracellular concentrations for each cation. We also show that, unlike our previously reported Cl--dependent cascade, which could use either GTP or ATP as kinase substrates, Na+ and K+ exert their differential regulatory effects on NDPK when ATP is the phosphate donor.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Collection of Human Respiratory Epithelial Cells

The subjects studied were all healthy young adults under the age of 45 yr undergoing surgery for reasons unrelated to nasal mucosal disease. Written informed consent and Tayside Committee on Medical Research Ethics approval were obtained. As detailed in Treharne et al. (31), immediately after anesthesia, respiratory epithelial cells were brushed from the inferior nasal turbinate epithelium with a cytology brush and dislodged into a nutrient medium (medium 199). Apically enriched membranes were then prepared. Briefly, a disrupted postnuclear supernatant was fractionated on a discontinuous sucrose gradient, and apical membranes were identified by alkaline phosphatase enrichment. Contaminating membranes were excluded by appropriate marker assays.

Phosphorylation of Membranes by Endogenous Kinase(s)

Apically enriched membrane pellets (made from pooled brushings, typically from 4 individuals) were resuspended in ice-cold 10 mM MOPS (pH 7.9 with KOH, final concentration of K+ 11 mM) containing 0.05% Triton X-100 (referred to as "membrane buffer"), with 20 µM dithiothreitol and 0.5% DMSO. Apical membrane proteins were incubated with various 10 mM cation-Cl- combinations (20 min at 4°C), followed by the addition of [gamma -32P]ATP for 5 min at 37°C. For the cation control experiments, MOPS buffer (pH 7.9) was made using either tetramethylammonium (TMA) hydroxide (final concentration 13 mM) or a mixture of KOH and NaOH (final concentrations of 4 and 13 mM, respectively). For the dose response studies (0-100 mM cation-Cl-), 10-µg aliquots of apically enriched membranes were incubated with 37 kBq of either [gamma -32P]ATP or [gamma -32P]GTP (final concentration 7 nM) at 37 or 4°C for the appropriate time and conditions. In all experiments, 1 µl of [gamma -32P]ATP or [gamma -32P]GTP (within 1 half-life of activity date) was spotted onto the side of the tube, and the reaction was started by a rapid spin to mix reagents. Phosphorylation was terminated by addition of 5× Laemmli sample buffer (16), followed by rapid mixing.

Quantitation of Phosphorylation

Proteins were separated by SDS-PAGE using 12.5% polyacrylamide gels on a Protean II slab cell (Bio-Rad). Prestained molecular mass markers were used to avoid the loss of phosphohistidine in the acid environment of staining and destaining of gels before quantification. The incorporation of phosphate into individual protein bands was quantified using electronic autoradiography (Canberra-Packard Instant Imager).

Method 1: normalizing against the cation inducing maximal phosphorylation. Method 1 is described in Ref. 30. Briefly, ordinate data (see Fig. 1) are means ± SE of percent net maxima. The percent net maximum for a given protein is calculated to compensate for the variation in intensity of phosphorylation between pooled membrane samples from different individuals. Data were normalized to the maximally phosphorylated band for each experiment, minus background. Background phosphorylation was defined as an area of the gel adjacent to the protein of interest but containing no phosphorylated proteins. Typically, apical membranes were bathed in a variety of cations. Thus, for a given protein (e.g., p19h), when the intensity of its phosphorylation was measured, the p19 lane with the greatest incorporated counts/min (cpm) was first assigned a value of 100%, and the rank order of the remainder were expressed relative to this value (%net maximum).

Method 2: normalizing phosphorylation relative to buffer control. During the dose-response studies, the object was to compare relative phosphate incorporation in the presence of Na+, K+, or NH+4. Thus all cpm incorporated into a given protein in the presence of buffer control were given the arbitrary value of 1 and the other cations were compared against this reference.

Immunoprecipitation of NDPK

Immunoprecipitation of NDPK has been described recently (21).

Chemical Reagents

All chemicals were of analytical grade and were purchased from Sigma or BDH (Poole, UK) except for the following: [gamma -32P]ATP and [gamma -32P]GTP were from NEN DuPont (Stevenage, UK), the acrylamide and other electrophoresis materials were from Bio-Rad, PBS was from Microgen Bioproducts, okadaic acid was from Calbiochem (Nottingham, UK), medium 199 was from Flow Laboratories, and NDPK antibodies were from Santa Cruz Biotechnology.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cations Regulate Phosphorylation of p19h and p37h

Figure 1 shows the results of bathing apically enriched human airway membranes in one of the Cl- salts of eight different cations (10 mM; Na+, K+, NH+4, Rb+, Li+, choline, tetraethylammonium, or TMA) before the initiation of phosphorylation of apical membrane proteins. The electronic autoradiograph (Fig. 1A) shows that the intensity of phosphorylation of a pair of 19- and 21-kDa proteins (p19h and p21h) and p37h is differentially dependent on the cation species bathing the membrane. We have recently shown that in sheep tracheal epithelium, p19s and p21s are isoforms of NDPK located within apically enriched membranes (21). We found that p21s was more sensitive than p19s to dephosphorylation by Mg2+, thus making it difficult to study because trace amounts of Mg2+ are essential for kinase activity. In the present study, p21h phosphorylation was so faint that it could not be accurately discriminated against background. Thus p19h phosphorylation was quantitated and showed significantly enhanced phosphorylation with Rb+, choline, and NH+4 (Fig. 1C) compared with 10 mM Na+. A similar cation-dependent pattern was seen for p37h, which was maximally phosphorylated with Rb+ (Fig. 1B). The two key differences between these phosphoproteins were that for p37h the presence of Na+ resulted in the least phosphorylation compared with all the remaining cations and, second, that p37h was more intensely labeled than p19h.


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Fig. 1.   Inhibitory effects of Na+ on phosphorylation of apical membrane proteins. A: electronic autoradiographic image shows differential effects of 8 cation-Cl- combinations (10 mM, each with 11 mM K+ in buffer) on 37-kDa phosphoprotein p37h and smaller 19- and 21-kDa phosphoprotein pair p19h and p21h. B and C: quantitation of p37h (B) and p19h (C). Ordinate (%net maximum phosphorylated species; means ± SE; n = 8 experiments; see MATERIALS AND METHODS) shows that 10 mM NaCl induced least phosphorylation of p37h, whereas incubation with all remaining cations was significantly enhanced (maximum with RbCl). In contrast, for p19h, phosphorylation was significantly enhanced relative to NaCl in presence of Rb+, NH+4, and choline (Chol) salts. *** P < 0.001; ** P < 0.01; * P < 0.05 compared with NaCl for p19h and p37h. Phosphoproteins visualized at ~30-kDa were observed in ~10% of gels, and their phosphorylation with different cations showed a similar profile to that of p19h. TEA, tetraethylammonium; TMA, tetramethylammonium; cpm, counts/min.

Identification of p21h as NDPK

Recently, we have shown that apical membranes derived from sheep airway provide a good model for Cl--dependent human airway phosphorylation (21). In that study, we used immunoprecipitation to identify p21s as an isoform of NDPK. The native form of NDPK is a hexameric protein made up of different combinations of 19- and 21-kDa proteins (37). Commercial antibodies are only available for the larger 21-kDa isoform (known in an alternative nomenclature as nm23-H1). Qualitatively, p21h phosphorylation mirrored that of p19h (see Figs. 1 and 3-5; see also Ref. 21). We therefore tested the idea that p21h was human nm23-H1 NDPK using polyclonal antibodies raised against the nm23-H1 human NDPK. Figure 2A shows the starting material, i.e., the phosphoproteins within a 20-µg aliquot from the phosphorylated membranes used for the immunoprecipitation. Figure 2B shows that a single phosphorylated band of 21 kDa was present in an aliquot from the final immunoprecipitate. Prior incubation of the anti-nm23-H1 antibody with the peptide against which it was raised eliminated the immunoprecipitation of this 21-kDa phosphoprotein (Fig. 2C). In further controls (data not shown), we could not immunoprecipitate any phosphorylated proteins using an unrelated antibody to the common antigen of the beta -subunits from G proteins. Having identified the 21-kDa isoform of NDPK, we increased the concentration of both the membranes and the antibody (10-fold) and were able to immunoprecipitate a phosphoprotein of 19 kDa in addition to p21 (Fig. 2D). A similar doublet was immunoprecipitated from a postnuclear supernatant of the H441 airway cell line (data not shown). These data, together with the evidence in Muimo et al. (21), suggest that NDPK is present in our phosphorylation cascade. We investigated the effects of Na+ and K+ concentration on the phosphorylation of the p19h-NDPK.


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Fig. 2.   Immunoprecipitation of phosphorylated p21h-nucleoside diphosphate kinase (NDPK). Apical membranes (200 µg protein) in 50 µl of membrane buffer were phosphorylated as described in MATERIALS AND METHODS. A: 10-µl aliquot was removed, run on 12.5% SDS-PAGE gel, and autoradiographed. B: remainder was immunoprecipitated (21) with 10 µl of an anti-nm23-H1 antibody. An autoradiograph of a 20-µl aliquot from final immunoprecipitate reveals a single phosphorylated 21-kDa band. C: same experiment was repeated using antibody preincubated with nm23 peptide epitope against which it was raised. D: coimmunoprecipitation of 19-kDa band with 10-fold increase in antibody concentration.

Na+ and K+ Have Opposite Effects on p19h-NDPK Phosphorylation

Comparison of the electronic autoradiographs in Figs. 3A and 4A shows that the phosphorylation of p19h-NDPK declines as NaCl concentration ([NaCl]) increases (Fig. 3), whereas the reverse is seen when [KCl] increases (Fig. 4). Two quantitative aspects of the cation dependence of the phosphorylation of p19h-NDPK are described (Figs. 3C and 4C). First, a quasi-exponential, [NaCl]-dependent decline in phosphorylation is present (EC50 10 mM). Second, the pattern for [KCl] shows the exact opposite: a concomitant increase in phosphorylation of p19h-NDPK (EC50 10 mM for added K+; see Buffer Controls and Inhibitory Effects of Na+). It is noteworthy that as phosphorylation reached its nadir (by increase of [NaCl] from 0 to 25 mM), p19h-NDPK phosphorylation approached its maximum with the addition of 25 mM [KCl]. The decline in phosphorylation induced by NaCl was a Na+-specific effect, because it could not be reproduced with NH4Cl, the latter mimicking K+ and showing an increase in the phosphorylation of p19h-NDPK (Fig. 4C).


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Fig. 3.   NaCl inhibits both p37h and p19h-NDPK phosphorylation. Membranes were preincubated with increasing concentrations of NaCl ([NaCl]; 0-100 mM) before phosphorylation with [gamma -32P]ATP. A: autoradiograph of SDS-PAGE gel showing rapid decline in phosphorylation of p37h and p19h as [NaCl] increases. B and C: quantitation showed an exponential decline in phosphorylation of both p37h (B) and p19h (C).


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Fig. 4.   KCl inhibits p37h (but not p19h-NDPK) phosphorylation. Experiment as in Fig. 3 but with increasing [KCl] or [NH4Cl] (0-100 mM). A: autoradiograph. B and C: quantitation of p37h (B) and p19h (C). In contrast to inhibitory effects of NaCl, both replacement cations augment p19-NDPK phosphorylation. However, cation-dependent inhibition of p37h is retained, albeit at a slower rate of decline. Data are means ± SE (n = 6 experiments).

Na+ and K+ Have Similar Effects on p37h Phosphorylation

Figure 3B shows that 50 mM NaCl induces a fivefold reduction in p37h phosphorylation compared with buffer control. Because the results for p19h-NDPK suggested that K+ and Na+ acted reciprocally, we used p37h phosphorylation to test the generality (or otherwise) of our earlier observations. The gel in Fig. 4A shows that increasing [KCl] or [NH4Cl] results in a decline in p37h phosphorylation irrespective of the cation-Cl- combination. This indicates that p37h phosphorylation is regulated differently from p19h-NDPK phosphorylation. Interestingly, the pattern of decline is also different (compare Figs. 3B and 4B). As [KCl] increases, there is a quasi-linear decline in p37h phosphorylation (EC50 25 mM) compared with the exponential fall seen with NaCl (EC50 10 mM; Fig. 3). KCl-dependent phosphorylation of p37h continues its linear descent beyond 50 mM, whereas for NaCl there was no significant decline after 50 mM.

Buffer Controls and Inhibitory Effects of Na+

The MOPS acid buffers were neutralized with KOH before the addition of membranes and therefore already contained an initial concentration of 11 mM K+ before the 10 mM cation-Cl- combinations were added. It remained possible that this excess of K+ was falsely enhancing p37h and p19h phosphorylation relative to Na+. However, replacement of the K+ with TMA or a combination of KOH and NaOH had no effect on the Na+-dependent decline in p37h and p19h phosphorylation (Fig. 5, A and B). Once again, the presence of Na+ resulted in the least membrane protein phosphorylation. We have reported previously (30) that the effects of increasing salt concentration were not due to the colligative properties of the phosphorylation buffer, because they were not reproduced with mannitol or sucrose. We concluded that Na+ was an important inhibitory influence on both p37h and p19h-NDPK phosphorylation, whereas K+ (and other cations) were stimulatory to NDPK alone.


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Fig. 5.   Na+ inhibition of NDPK phosphorylation occurs irrespective of cation used in membrane buffer. Membranes were preincubated in different buffers with increasing [NaCl] (0-100 mM) before phosphorylation with [gamma -32P]ATP. A: quantitation of p37h phosphorylation with buffer neutralized with KOH, TMA hydroxide (TMAOH), or KOH-NaOH (see MATERIALS AND METHODS). Phosphorylation was inhibited with increasing NaCl under all 3 conditions. B: equivalent data for NDPK phosphorylation show that replacement of K+ did not affect Na+ inhibition of NDPK phosphorylation (data for 1 experiment shown, n = 2).

Low Temperature Eliminates Na+ Inhibition of NDPK Phosphorylation

The cation-dependent changes in phosphorylation could have been mediated via kinase(s), phosphatase(s), or both. All the results described so far relate to steady-state experiments (phosphorylation for 5 min at 37°C) in which both sets of enzymes were (potentially) active. At 4°C kinases have been reported to retain their activity relative to phosphatases (3), providing a simple method of discriminating between their relative contributions. To define which reaction conferred generation of cation sensitivity, we repeated the NaCl and KCl dose responses at the lower temperature. Figure 6 shows the typical effects of increasing [NaCl] or [KCl] at 4°C and reveals five interesting results: 1) p37h is no longer phosphorylated; 2) p19h and p21h are the sole phosphorylated species, probably reflecting autophosphorylation of NDPK (21); 3) Na+ and K+ now have similar effects on the phosphorylation of p19h-NDPK (compare with Figs. 3 and 4); 4) lowering the reaction temperature generates a new profile of salt-dependent phosphorylation that differs from that seen at 37°C with either cation-Cl- combination: at 4°C, p19h phosphorylation declines with either cation (contrast result at 37°C; 25 mM NaCl was selectively inhibitory; see Fig. 3); and 5) phosphorylated p21h is measurable for the first time and mirrors the profile of p19h, albeit at a lower intensity (quantitative data not shown). We conclude that at 4°C discrimination between Na+- and K+-dependent phosphorylation is absent. This discrimination in phosphorylation at 37°C with Na+ suggests the presence of a Na+-sensitive phosphatase(s).


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Fig. 6.   Temperature reduction eliminates Na+ selectivity and alters profile of NDPK phosphorylation. Representative autoradiographic profile (n = 3) using phosphorylation protocol as described in Figs. 3 and 4 except that temperature was reduced to 4°C in presence of either NaCl (A and C) or KCl (B and D). A and B: autoradiographs. C and D: quantitation. At 4°C, p37h is no longer phosphorylated, but p21h is phosphorylated, and its profile mirrors that of p19h. Both proteins are phosphorylated at salt concentrations below 25 mM but extensively dephosphorylated above this threshold. K+-selective stimulation of p19h-NDPK phosphorylation is not observed at 4°C.

Role of Phosphatases

Our previous data showed that the kinases in this system were insensitive to conventional inhibitors (30), and we implicated phosphatase involvement because of the loss of Cl--dependent phosphorylation when the phosphatase-resistant analog adenosine 5'-O-(3-thiotriphosphate) was substituted for ATP (31). The conventional approach to understanding the role of phosphatases is to add specific inhibitors of known phosphatases. This approach was not successful because no differences in phosphorylation were observed when experiments were conducted in the presence or absence of okadaic acid (Fig. 7). Microcystin LR and calyculin A were equally ineffective (data not shown). To show that the relationship between the putative phosphatase(s) and Na+ was not due to an anion, we incubated membranes in okadaic acid (1 µM) and then added two different Na+-anion combinations: NaCl or sodium gluconate. We used 40 mM Na+ (fourfold above EC50) and found that, irrespective of the accompanying anion, okadaic acid failed to restore (Na+-inhibited) phosphorylation of p19h (Fig. 7), confirming our earlier observations that Na+ was not activating protein phosphatase 1 or 2A (PP-1 or PP-2A) to induce a decline in p19h phosphorylation. This experiment also revealed an unexpected result with respect to the species of anion accompanying the Na+. In the presence of sodium gluconate, p19h phosphorylation was significantly elevated compared with NaCl, suggesting that 1) the species of anion influences p19h dephosphorylation and 2) a synergy exists between Na+ and Cl- in the dephosphorylation of NDPK. The latter idea has resonance with our earlier data (see Fig. 2 of Ref. 30) showing that gluconate and Cl- lie at opposite ends of the phosphorylation spectrum when other phosphoproteins within this apical cascade of phosphorylation are studied. Although these data are consistent with a phosphatase-mediated Na+-activated dephosphorylation of NDPK, Na+-dependent inhibition of the kinase could still have occurred. We excluded this possibility by measuring initial (quasi-zero time) incorporation of phosphate into NDPK in the presence or absence of Na+. Membranes were incubated with [gamma -32P]ATP on ice for 10 s in 10 mM NaCl. We found that the cpm incorporated into NDPK in the presence of Na+ exceeded buffer control by an average factor of 2 (mean ± range 651 ± 153 vs. 281 ± 113 cpm in the presence and absence of Na+, respectively), thus excluding the notion of an inhibitory Na+-kinase interaction.


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Fig. 7.   Na+-dependent dephosphorylation is modulated by anion and is insensitive to okadaic acid (OA). Paired membranes were preincubated at 4°C with NaCl or sodium gluconate (40 mM each) or with buffer alone for 20 min. Okadaic acid (1 µM) was added to 1 set for a further 20 min before phosphorylation with [gamma -32P]ATP for 5 min at 37°C. A: autoradiograph of SDS-PAGE. B: quantitation (n = 3). Phosphorylated p19h was abolished when NaCl was present. However, when NaCl was replaced with sodium gluconate, inhibition was lost and p19h showed a 55% reduction in phosphate incorporation compared with control. Okadaic acid could neither restore phosphorylation state of p19h-NDPK with NaCl nor enhance that with sodium gluconate.

Nucleotide Substitution Eliminates Na+ Inhibition of Phosphorylation

Our previous results had shown that the Cl- sensitivity of p37h phosphorylation occurred principally but not exclusively (depending on the cation-anion combination) when [gamma -32P]GTP was the kinase substrate. We therefore tested the effects of this guanine nucleotide on the above profile of phosphorylation. Apical membrane proteins were phosphorylated for 5 min at 37°C with [gamma -32P]GTP instead of [gamma -32P]ATP in the presence of increasing concentrations of NaCl, KCl, or NH4Cl. Quantitation of p37h phosphorylation from ATP and GTP is shown in Fig. 8, A and B, respectively: in the presence of [gamma -32P]GTP, NaCl induces a quasi-linear decline in phosphorylation of p37h. We observe that p37h phosphorylation now decreases in a similar manner, irrespective of the cation species (Fig. 8; compare A and B). In addition, for p19h-NDPK phosphorylation from [gamma -32P]GTP, Na+ now has two different effects. First, no inhibition of phosphorylation occurs, and, second, a slow increase in phosphorylation occurs as NaCl approaches 100 mM (probably Cl- mediated; see Fig. 1 of Ref. 21).


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Fig. 8.   Na+ inhibits NDPK phosphorylation by ATP and not GTP pathway. Effects of increasing cation-Cl- concentration (0-100 mM) in presence of either [gamma -32P]ATP (A and C) or [gamma -32P]GTP (B and D) as phosphate donors. A and B: p37h. C and D: p19h. Data are expressed relative to cpm incorporation into buffer control (given an ordinate value of 1; see MATERIALS AND METHODS). Comparison of C and D shows that differential effects of Na+ and K+ on p19h only occur via ATP pathway. In presence of ATP, there is a 4-fold excess incorporation of phosphate into p19h. In contrast, for p37h, all cation-Cl combinations inhibit phosphorylation, irrespective of nucleotide. For ease of comparison, data from Figs. 3 and 4 are replotted (A) and show that Na+ induces steepest decline in p37h phosphorylation with ATP (but not GTP) as phosphate donor.

The combined evidence suggests that the Na+-dependent regulation of phosphorylation, in a range that is likely to be present in the intracellular environment (0-25 mM Na+), only occurs when ATP is present as the kinase substrate. This is further illustrated by quantitating the phosphorylation of p19h with [gamma -32P]ATP (Fig. 8C). The phosphate incorporation into p19h-NDPK shows (relative to buffer control) a fourfold excess in the presence of K+. In contrast, there is a 50% decline in phosphorylation with Na+. NH+4 occupies an intermediate position. However, in the presence of [gamma -32P]GTP, p19h-NDPK cannot discriminate between cation species. We also find that p37h is inhibited by either cation (Na+ > K+), suggesting Na+-dependent dephosphorylation is specific to NDPK.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Control of Vectorial Ion Transport in Epithelia

Ion substitution is a general stratagem for the study of ion transport across cell membranes. Our study suggests that ion substitution is not a neutral process where the Na+-absorptive apical membrane of airway epithelium is concerned. Apical membranes from this epithelium experience a high-K+ cytosolic environment and yet regulate the local rise in Na+ concentration during Na+ absorption while maintaining a low [Na+]i. This membrane displays a complex interaction (Refs. 21 and 30 and this study) between ion species and concentration, nucleotides, and the phosphorylation state of NDPK, a kinase known to regulate K+ channels (10, 38), secretion (19), and cellular energy metabolism (35). We now show that membrane-bound NDPK discriminates between K+ and Na+ at intracellular concentrations physiologically relevant for each cation in the presence of ATP but not GTP. This is contrary to the literature, which reports that purified NDPK does not discriminate between nucleotides (22, 23, 37). We show that Na+ reduces the net phosphorylation of membrane-bound NDPK in the presence of ATP alone. This difference in phosphorylation between ATP and GTP may be explained either by NDPK being phosphorylated on different sites with each nucleotide or by a GTP-mediated inhibition of the phosphatase(s) targeting the kinase.

Consequences of Ion Regulation of NDPK

In epithelia, it is well recognized that the opening of basolaterally located K+ channels provides the driving force for secretion via the apical membrane (17). At the apical membrane, UTP is an important Cl- secretagogue (11, 29), and phosphorylated NDPK provides the sole pathway for UTP synthesis (from UDP and ATP). We now find that the anion (Cl- >> gluconate) accompanying Na+ modulates the degree of inhibition of NDPK phosphorylation. Because we know that a [Cl-]i of >40 mM enhances NDPK phosphorylation (21), our results provide a plausible feedback loop between UTP synthesis and [Na+]i and [Cl-]i. Specifically, as [Na+]i rises, NDPK phosphorylation falls, but in the presence of a Na+ substitute [N-methyl-D-glucammonium (NMDG), K+, or NH+4] NDPK phosphorylation does not decline. Our data predict that a high [Na+]i will be inhibitory to UTP-mediated processes in human airway because Na+ overcomes the enhancing effect of KCl on NDPK phosphorylation.

Multiple Functions of NDPK in Other Systems

By an unknown mechanism, NDPK regulates ACh-activated K+ channels (KACh channels) (38). When a muscarinic agonist is present, KACh channels open, and antibodies to NDPK inhibited this opening. However, simultaneous suppression of GTP production via NDPK does not prevent kinase-mediated channel inhibition. This dissociation between the nucleotide synthetic function of NDPK and its role as a conventional kinase is well recognized but poorly understood (9, 19, 35). The same study (38) also found that inhibition of NDPK had no effect on baseline KACh channel activity in the absence of agonist, suggesting that channel modulation by NDPK is a "poststimulus" event. It is precisely under these conditions that intracellular ion concentrations change rapidly. The recognized functions of this kinase, regulation of pancreatic secretion and cellular energy metabolism, are compatible with an integrative role for NDPK in membrane channel function via ions as second messengers. Our current and previous data with anions (21, 30) point to a phospho-relay, which is ideally suited to respond to ionic perturbations in epithelia. NDPK is ideally positioned to sense ions on the cell surface (33), on the inner leaflet of the plasma membrane (12), and in the cytoplasm (22). The concept of an ion sensor is particularly relevant to human nasal airway epithelium, which is subject to a wide variety of environmental challenges (temperature, heat, relative humidity, and so forth) with a potential to disturb the tonicity and/or composition of the airway surface liquid bathing the cilia and thereby the composition of the epithelial cytosol (17, 30). Despite these potential perturbations, [Na+]i is held constant at ~10-fold below its plasma value (25, 26), precisely in the range of maximum Na+ sensitivity of NDPK phosphorylation.

Na+ is unlikely to inhibit NDPK phosphorylation directly, because our "initial rate experiments" show that phosphate incorporation into the kinase is not reduced in the presence of 10 mM NaCl. Indeed, the observed twofold elevation of phosphate incorporation with Na+ over buffer control is consistent with our proposal that a Na+-activated phosphatase is present. This novel notion is supported by the loss of Na+ sensitivity at 4°C, a temperature reported to inhibit phosphatase activity (Ref. 3, see also Ref. 28). Although the phosphatase is unknown, our data suggest that it cannot be PP-1 or PP-2A (okadaic acid has no effect) and is unlikely to be PP-2B or PP-2C (ovine airway data, Ref. 21). A 10-kDa Na+-dependent phosphatase from bacteria with maximal in vitro activity directed against human NDPK has been described (27). This phosphatase has very little activity in the presence of K+, compatible with our data.

Cation-Anion Interactions With Apical Membrane Phosphoproteins

The Na+-dependent inhibition of NDPK phosphorylation was less marked when the accompanying anion was switched from Cl- to gluconate. This anion dependence is reminiscent of our previously characterized Cl--dependent protein p37h, whose phosphorylation was enhanced by gluconate compared with Cl-, albeit in the presence of GTP (and not ATP; note the contrast with the current results). The combined data suggest that anion and cation differentially regulate NDPK and p37h phosphorylation using different nucleotides. However, we do not understand the differences in the pattern of Cl- dependence between NDPK and p37h. In the present study, p37h phosphorylation shows no difference in sensitivity between Na+ and K+ with either ATP or GTP. Nevertheless, the anion-cation combination is important for the net phosphorylation of p37h. For example, a dose response with NMDG chloride induces a concentration-dependent peak of phosphorylation (~40 mM), whereas NaCl induces an exponential decline and KCl induces a quasi-linear decline.

Although the NDPK phosphorylation reported in this paper is likely to be autophosphorylation, it remains possible that another Na+-sensitive kinase phosphorylates NDPK. Casein kinase 2 (CK2)-dependent phosphorylation of serine adjacent to the phosphohistidine site in NDPK is known to prevent NDPK autophosphorylation (2, 8). That 100 mM NaCl promotes CK2 activity in vitro is consistent with the second kinase hypothesis. Consequently, in this model, Na+ could activate CK2 to inhibit NDPK phosphorylation indirectly. However, such a model could not readily explain why a reduction in temperature eliminates Na+-dependent inhibition, since kinase activity is not likely to be curtailed. We are not aware of data on the temperature dependence of CK2 activity.

NDPK as an Integrator of [Na+]i, [K+]i, and [Cl-]i

Both cation and anion concentrations play regulatory roles in epithelial function that are poorly understood. There is a direct link between [Cl-]i and Cl- conductance through the cystic fibrosis transmembrane conductance regulator (36). Thus Cl- regulates Cl- exit. Furthermore, [Cl-]i regulates Cl- entry via the Na+-K+-2Cl- cotransporter (18) and additionally controls cation transport (6). Dinudom et al. (6) showed that as [Cl-]i increased, the inward Na+ current decreased. Simultaneously, the inward Cl- current increased. In rat fetal distal lung epithelium, Marunaka et al. (20), studying Na+ absorption, found that activation of a nonspecific cation channel depended on [Cl-]i. Thus there appears to be a relationship between the identity and concentrations of intracellular ions and their rates of ion transport, but the links are poorly understood. Dinudom et al. (5) reported that the [Cl-]i regulates Na+ transport via a G protein. However, further complexity is introduced by the known Cl- sensitivity of some G proteins (see Ref. 21 for discussion). NDPK associates with G proteins, but the functional consequences of this binding are controversial. It has been suggested that NDPK and Gs may coexist such that the complex could allow the localized formation and exchange of GTP for GDP bound to the G protein (13). Randazzo et al. (24) rebutted this concept but used a model system that did not control for the presence of Na+ (their buffers were based on Na+-neutralized HEPES). Interestingly, it was the use of such buffers that led us to the preliminary observations on the inhibitory effects of Na+ in our system. The above authors concluded that, since they could find no evidence for a role of NDPK in GDP-to-GTP conversion when the GDP was bound to G proteins, the role of NDPK as a local GTP generator was untenable. Our data suggest that changes to the ionic composition of the buffer may alter these results. Alternatively, the kinase function of NDPK could operate despite inhibition of its phosphotransferase role and regulate G proteins by a different mechanism (see above and Ref. 9).

The airway epithelium both secretes and absorbs fluid, but it is unclear whether or not both processes occur in a single cell within the same time frame. Our work points to a molecular switch between these processes regulated by ion-dependent phosphorylation. We propose the following model (Fig. 9). When [Cl-]i is high and [Na+]i is <10 mM, NDPK is phosphorylated (on one or more sites). Assuming this phospho-NDPK is essential for function (UTP synthesis and K+ channel opening, for example), we predict that as K+ leaves the cell, the resultant membrane hyperpolarization "coupled" to the synthesis of UTP will drive apical Cl- secretion. However, the mechanism that might transport this UTP to the airway lumen is unclear. The resultant fall in [Cl-]i will turn off the kinase as well as promote KCl entry via the Na+-K+-2Cl- cotransporter, completing the feedback loop. Should cAMP-activated apical Na+ absorption be occurring, the membrane-delimited rise in [Na+]i will itself turn off NDPK via phosphatase activity. The latter predicts that a high [Na+]i turns off UTP production. Finally, our unusual finding with respect to the anion accompanying the Na+ suggests a two-site model for ion binding within this cascade.


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Fig. 9.   Ion sensing via a membrane-delimited phospho-relay. Cartoon shows proteins bound to inner leaflet of apical membrane. ATP-phosphorylated NDPK acts both as a phosphotransferase (making UTP) and as a regulator of K+ channels (activated during ACh-stimulated epithelial secretion). When Cl- entry (e.g., via Na+-K+-2Cl- cotransporter) drives intracellular Cl- concentration ([Cl-]i) above 40 mM, phospho-NDPK is promoted. Mechanism is unclear, and different phosphorylated forms of NDPK may occur as [Cl-]i changes. Rise in [Cl-]i inhibits Cl- entry (not shown). Conversely, a rise in [Na+]i activates a phosphatase and deactivates NDPK, but the anion accompanying rise in [Na+]i modulates degree of deactivation (not shown; see RESULTS). KACh, ACh-activated K+ channels; PP'ase, protein phosphatase; Cl-i, intracellular Cl-; K+i, intracellular K+.

    ACKNOWLEDGEMENTS

We thank Anna Crichton for collecting nasal brushings and the Ninewells theater staff for help over many years.

    FOOTNOTES

L. J. Marshall is a Wellcome Prize Student, R. Muimo was supported by the Wellcome Trust, and C. E. Riemen was supported by the United Kingdom Cystic Fibrosis Trust. BioMed II Grant BMH4-CT96-0602 and Tenovus (Scotland) provided support and purchased equipment; the Anonymous Trust provided ongoing support.

Address for reprint requests: A. Mehta, Dept. of Child Health, Ninewells Hospital Medical School, University of Dundee, Dundee DD1 9SY, UK.

Received 3 December 1997; accepted in final form 30 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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