Department of Child Health, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom
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ABSTRACT |
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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
[-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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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-ClQuantitation 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: [ ![]() |
RESULTS |
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Cations Regulate Phosphorylation of p19h and p37h
Figure 1 shows the results of bathing apically enriched human airway membranes in one of the Cl
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Identification of p21h as NDPK
Recently, we have shown that apical membranes derived from sheep airway provide a good model for Cl
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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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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-ClBuffer 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
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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
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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
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Nucleotide Substitution Eliminates Na+ Inhibition of Phosphorylation
Our previous results had shown that the Cl
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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
[-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
[
-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.
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DISCUSSION |
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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 ClMultiple 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 ClAlthough 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
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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ACKNOWLEDGEMENTS |
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We thank Anna Crichton for collecting nasal brushings and the Ninewells theater staff for help over many years.
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FOOTNOTES |
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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.
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