Departments of Chemistry and Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
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ABSTRACT |
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We examined protein kinase C (PKC)-dependent regulation of Na+-K+-ATPase in frog mucociliary cells. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (diC8) either in intact cells or isolated membranes resulted in a specific inhibition of Na+-K+-ATPase activity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximal inhibition concentration (IC50) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA and diC8] and were not influenced by Ca2+. Analysis of the ouabain inhibition pattern revealed the presence of two Na+-K+-ATPase isoforms with IC50 values for cardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM, respectively. Most importantly, the isoform possessing a higher affinity for ouabain was almost completely inhibited by TPA, whereas its counterpart was hardly sensitive to the PKC activator. The results suggest that, in frog mucociliary cells, PKC regulates Na+-K+-ATPase and that this action is related to the specific Na+-K+-ATPase isoform.
sodium-potassium-adenosinetriphosphatase isoforms; phorbol esters; ouabain
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INTRODUCTION |
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IT HAS BEEN ESTABLISHED that ciliary activity of epithelial cells can be regulated by hormonal stimuli (32). Recently, we have shown that activation of protein kinase C (PKC) enhances ciliary beating in tissue cultures from frog esophagus. In addition, we observed the involvement of PKC in propagation of ciliary activity stimulated by purinergic agonists (21). Moreover, it was found that these physiological responses are associated with PKC-induced Ca2+ influx (21), which similar to potassium fluxes (21, 32) may play an important role in ciliary stimulation. The nature of PKC-induced Ca2+ influx remains unclear, although we have determined that it is not blocked by verapamil at concentrations appropriate for voltage-dependent Ca2+ channel blockage (21). These facts suggest that PKC activates Ca2+ influx via a mechanism that does not involve voltage-operated Ca2+ channels. One of the possible routes for Ca2+ entry may be provided by Na+/Ca2+ exchange, which uses energy of Na+ gradient generated by an electrogenic Na+ pump. Thus inhibition of the Na+ pump may increase Ca2+ influx via Na+/ Ca2+ exchange.
The Na+ pump (Na+-K+-ATPase) is responsible for the transport of solutes and water across the epithelium (5, 29). In both epithelial and nonepithelial cells, PKC was shown to be involved in short-term modulation of enzymatic and ion-transporting activity of Na+-K+-ATPase by various hormones, neurotransmitters, and growth factors (5, 14). Thus activation of PKC mediated by different receptor agonists or phorbol esters was found to stimulate Na+-K+-ATPase in rat brain capillary endothelium (19) or transformed human nonpigmented ciliary epithelial cells (24) but inhibited its activity in choroid plexus (17), rabbit nonpigmented ciliary epithelial cells (11), rat proximal and distal nephron (3, 30), and opossum kidney cells (23).
We undertook the present study to elucidate whether PKC activation influences Na+-K+-ATPase activity in frog esophagus mucociliary cells. To date, Na+-K+-ATPase has not been characterized in mucociliary cells. We show that activation of PKC in either intact mucociliary cells or isolated membranes results in a specific inhibition of Na+-K+-ATPase activity. Our data implicate the Na+-K+-ATPase isoform with a very high affinity for the cardiac glycoside ouabain as a putative target for this regulatory response. Such isoform-specific regulation of Na+-K+-ATPase may play a physiological role in modulation of ciliary beating.
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MATERIALS AND METHODS |
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Materials.
12-O-tetradecanoylphorbol-13-acetate
(TPA), 1,2-dioctanoyl-sn-glycerol
(diC8), and staurosporine were obtained from Calbiochem-Novabiochem (La
Jolla, CA). Pyruvate kinase (PK)-lactate dehydrogenase (LDH) suspension, Na2-ATP, leupeptin,
and 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF)
were from Boehringer Mannheim (Mannheim, Germany).
Tris(hydroxymethyl)aminomethane (Tris), dithiothreitol (DTT),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), NADH, di(Tris)
p-nitrophenyl phosphate
(p-NPP), aprotinin, ouabain,
deoxyribonuclease II (DNase), sodium
phospho(enol)pyruvate, and
4
-phorbol were from Sigma (St. Louis, MO). Sucrose was from Merck
(Darmstadt, Germany). Protein assay kit was from Bio-Rad (Richmond,
CA). Alamethicin, containing peptide F30 as the major component, was a
generous gift of Dr. V. B. Ritov (Moscow State University, Moscow,
Russia).
Tissue preparation. Frog esophagi were isolated using the procedure described previously (12). Briefly, frogs (Rana ridibunda) were killed by pithing the brain and spinal cord. The esophagus was removed and washed of mucus and blood in an ice-cold Ringer solution containing (in mM) 120 NaCl, 2.5 KCl, 1.8 CaCl2, 1.1 Na2HPO4, and 0.85 NaH2PO4, pH 7.2, and was stored in this buffer for <1 h until use.
Tissue treatment with PKC modulators and membrane preparation. Washed whole esophagi were preincubated in the Corning six-well tissue-culture plate (1 esophagus/well) in 5 ml Ringer solution at room temperature for 25 min. Where indicated, staurosporine (100 nM) was present during preincubation. TPA (80 nM) or diC8 (20 µg/ml) was then added for another 5 min. Control samples were incubated under the same conditions with no additions. The incubations were stopped by rapid cooling of the esophagi to 4°C as described (3) followed by three quick washes with an ice-cold Ringer solution. The mucociliary cell layer of each esophagus was scraped with a sharp blade into 1 ml of ice-cold buffer A containing (in mM) 20 Tris-Cl (pH 8), 2 MgCl2, 1 DTT, 1 EGTA, 0.5 AEBSF, and 250 sucrose supplemented with 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml DNase. Light microscopic examination of the cell suspension revealed that ~90% of cells were of ciliary origin. The suspension was homogenized on ice by 20 strokes in a 1-ml Dounce homogenizer, and the homogenate was centrifuged at 1,000 g for 15 min at 4°C. The pellet containing nuclei and cell debris was discarded, and the supernatant was centrifuged at 40,000 g for 1 h. The resulting membrane pellet was resuspended in ice-cold buffer B containing (in mM) 40 HEPES (pH adjusted to 7.8 with Tris base), 1 EGTA, and 250 sucrose and used immediately in the enzymatic assays.
Large-scale membrane preparation from nontreated esophagi.
The following procedures were performed at 4°C. Mucociliary cell
layers scraped from 10-12 nontreated esophagi were washed 3 times
in Ringer solution by centrifugation at 250 g for 5 min. Cells were then
homogenized in 20 ml buffer A by a
Polytron homogenizer (Kinematika, Lucerne, Switzerland) at setting 5, three times for 30 s each, with 1-min intervals. Nuclei and cell debris
were removed by centrifugation at 1,000 g for 10 min. Membranes were pelleted from the supernatant by high-speed centrifugation (40,000 g for 1 h) and resuspended in
buffer B as above. Aliquots of the
suspension were then frozen in liquid nitrogen and stored at
70°C until use.
ATPase assay. ATPase activity was determined by the enzyme-linked assay (27). Before assay, membranes were treated with the pore-forming antibiotic alamethicin (1 mg/mg protein; 40-80 µg/ml) in buffer B for 20 min at room temperature (10). This treatment resulted in about a twofold increase in Na+-K+-ATPase activity (data not shown), indicating that ~50% of membrane fragments in the preparations were tightly sealed vesicles. For total ATPase assay, membranes (1-2 µg protein) were then incubated with test compounds in 200 µl buffer C containing (in mM) 40 HEPES (pH adjusted to 7.4 with Tris base), 100 NaCl, 20 KCl, 3 MgCl2, 1 EGTA, 5 NaN3, 2 phospho(enol)pyruvate, 2.5 ATP, and 0.4 NADH and 4 U/ml each of PK and LDH at 37°C. Oxidation of NADH was registered continually at 340 nm for up to 40 min in a 96-well plate using a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA). In the preliminary experiments, these conditions were found to be optimal (data not shown). Ouabain-insensitive ATPase activity was measured accordingly in buffer C containing 1 mM ouabain. Na+-K+-ATPase activity was calculated as the difference between the correspondent linear sections of total and ouabain-insensitive ATPase activity curves.
p-Nitrophenylphosphatase assay. The partial reaction of Na+-K+-ATPase, K+-dependent p-nitrophenylphosphatase (K+-p-NPPase) activity was determined essentially as described previously (9) by measuring conversion of p-NPP to p-nitrophenol (p-NP). For total p-NPPase activity measurement, membranes (1-2 µg protein), pretreated with alamethicin as above, were incubated with or without test compounds in 100 µl buffer D containing (in mM) 40 HEPES (pH adjusted to 7.8 with Tris base), 5 MgCl2, 5 p-NPP, and 1 EGTA in the presence or absence of 20 mM KCl at 37°C. Production of p-NP was monitored continually for up to 40 min at 405 nm in a 96-well plate using a microplate reader. In some experiments, Na+- and ATP-activated p-NPPase activity (13) was measured in buffer D supplemented with 50 mM NaCl and 0.1 mM ATP in the presence or absence of a lower concentration (2 mM) of KCl. In both cases, K+-p-NPPase activity was calculated as the difference between the corresponding linear sections of total and K+-independent activity curves. K+ stimulation of this activity was completely inhibited by 1 mM ouabain.
Miscellaneous. Protein was determined by the method of Bradford (6) following sample incubation with 0.2% Triton X-100. Bovine serum albumin was used as a standard. Concentration of free Ca2+ in ATPase assays was varied by using Ca2+-EGTA buffers. Free Ca2+ concentration was calculated by a computer program (18), taking into account pH and concentrations of EGTA, ATP, Mg2+, and monovalent cations. Dose-dependence curves were analyzed using the KaleidaGraph program (Abelbeck Software) by fitting the data to one- to three-component models using a nonlinear least-squares analysis of the sum of general logistic functions.
Statistics. Data are presented as means ± SE. Statistical comparisons were made using Student's t-test. Results were considered statistically significant if P < 0.05.
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RESULTS |
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Effects of tissue treatment with PKC activators. Recently, we showed that activation of PKC in primary culture of frog esophagus mucociliary cells by TPA or the diacylglycerol analog diC8 results in a marked (2- to 3-fold) enhancement of ciliary beat frequency with a maximal stimulation at ~2-5 min (21). To examine whether Na+-K+-ATPase activity in these cells is affected under similar conditions, whole frog esophagi were incubated for 5 min with 80 nM TPA or 58 µM (20 µg/ml) diC8 (concentrations that produce a maximal effect on ciliary beating; Ref. 21). Na+-K+-ATPase was then assayed in membranes prepared from mucociliary cells. The enzyme activity was determined as its partial K+-p-NPPase reaction in buffer devoid of PKC activators and ATP to exclude the possibility of an additional PKC-dependent Na+-K+-ATPase modification during the assay. As shown in Fig. 1, tissue exposure to either TPA or diC8 caused a significant 25-35% decrease in K+-p-NPPase activity of mucociliary cell membranes as compared with the control. Inhibition was stable throughout a 30- to 40-min reaction time course (data not shown) and selective with respect to K+-p-NPPase, since neither of the two PKC activators influenced ouabain-insensitive p-NPPase activity (Fig. 1). The effects of both TPA and diC8 were abolished in the presence of the PKC inhibitor staurosporine (100 nM), which did not change K+-p-NPPase activity by itself (Fig. 1), indicating that these effects are mediated by PKC activation.
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Direct effects of PKC activators in isolated membranes.
To elucidate the mode of action of the PKC activators on
Na+-K+-ATPase,
we first examined whether these compounds directly affect ATPase and
p-NPPase activities in membranes
isolated from mucociliary cell layers of nontreated esophagi. TPA (Fig.
2A) or
diC8 (Fig. 2B) added to the ATPase
assay caused an appreciable (35-45%) concentration-dependent inhibition of ouabain-sensitive
Na+-K+-ATPase
activity with a half-maximal effect
(IC50) at 0.5 ± 0.1 nM and
2.4 ± 0.2 µM, respectively, whereas ouabain-insensitive ATPase
activity was not altered (Fig. 2, A
and B). At concentrations of
5-10 nM, TPA produced maximal inhibition of
Na+-K+-ATPase
activity. The inhibitory effects of both TPA (Fig.
3) and diC8 (data not shown) were time
dependent, reaching a steady-state level in ~10 min of the reaction
course. Involvement of PKC in Na+-K+-ATPase
inhibition was confirmed by the facts that 4-phorbol (a phorbol
ester that will not activate PKC) did not significantly influence
Na+-K+-ATPase
activity (Fig.
4A) and
that the inhibitory effects of both TPA and diC8 were abolished by
staurosporine (Fig. 4B), similar to
the action observed in whole cells (see Fig. 1).
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DISCUSSION |
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In the present study, we for the first time partially characterized Na+-K+-ATPase activity in frog esophagus mucociliary cell membranes and studied its responses to PKC activation both at the tissue and cell-free level.
Similar to data reported by others in several cell types (5, 11, 17,
23), we found that application of the PKC activators TPA and diC8 to
intact cells produced a specific inhibition of Na+-K+-ATPase
activity that could then be detected in isolated membranes (see Fig.
1). On the other hand, an important novel finding of our study is that
a similar or even stronger decrease in
Na+-K+-ATPase
activity was observed when PKC activators were added directly to the
membranes isolated from nontreated cells (see Figs. 1 and 2). This
suggests that the amount of PKC present in the mucociliary cell
membrane even in the absence of stimulation by TPA or diC8 (known to
induce membrane translocation of the cytosolic enzyme; Ref. 26) is
sufficient to mediate inhibition of
Na+-K+-ATPase
activity. Our preliminary experiments showed the presence of the
Ca2+-independent PKC -isozyme
in membrane preparations isolated from nonstimulated frog ciliary cells
(unpublished data), which might account for the
Ca2+-independent
Na+-K+-ATPase
inhibition by PKC activators (see Fig. 6).
Exogenous PKC has been shown to directly phosphorylate the -subunit
of
Na+-K+-ATPase
in purified enzyme preparations. Thr-15 and Ser-16 were detected to be
the PKC phosphorylation sites on the
1-subunit of
Bufo marinus enzyme (1), whereas
Ser-11 and Ser-18 were shown to be modified by PKC in the rat kidney
ATPase
-subunit (16). Phosphorylation by PKC failed to change
Na+-K+-ATPase
activity in rat kidney enzyme preparation (15), whereas it inhibited
the activity (by ~40-50%) of the shark rectal gland enzyme (4).
Exogenous PKC also inhibited
Na+-K+-ATPase
activity in partially purified preparations and basolateral membrane
vesicles from rat renal cortex (4). Phosphorylation of
Na+-K+-ATPase
by endogenous PKC was observed following treatment of intact cells with
various receptor agonists and PKC activators (17, 22, 23). This
phosphorylation was accompanied by a reduction in enzyme activity in
kidney epithelial cells (23) and in the choroid plexus (17). It has
been recently demonstrated that
2- and
3-subunits from different
species are also phosphorylated by PKC, although to a lower extent than
1 (2). Amphibian
(Bufo marinus and
Xenopus)
1-subunits can be
phosphorylated by PKC both in vivo and in vitro (2, 8). Phosphorylation
by PKC of the amphibian
2- and
3-subunits has not been
reported. Neither modulation of
Na+-K+-ATPase
activity nor its phosphorylation by endogenous PKC present in isolated
membranes has previously been demonstrated.
Inhibition of
Na+-K+-ATPase
by PKC activators in ciliary cell membranes might also be associated
with phosphorylation of the Na+-K+-ATPase
-subunit. Indirect evidence for the role of protein phosphorylation in this process was obtained by measuring the partial reaction of
Na+-K+-ATPase
(K+-p-NPPase
activity) using a non-energy-rich phosphate donor
(p-NPP). Neither TPA nor diC8
influenced
K+-p-NPPase
activity in the absence of ATP when added to the membranes isolated
from the nontreated tissue. However, the inhibitory effect was restored
upon addition of the nucleotide to the assay (Fig. 5,
A and
B). This is in contrast to the
reduction in
K+-p-NPPase
activity seen even in the absence of ATP with membranes prepared from
TPA- or diC8-exposed cells (see Fig. 1). The latter finding suggests
that stimulation of PKC in whole cells may result in a stable
modification of
Na+-K+-ATPase,
which can still be detected in isolated membranes by measuring its
ATP-independent partial phosphatase reaction. Such modification may
also be caused by mechanisms other than direct phosphorylation of the
Na+-K+-ATPase
-subunit. These mechanisms may include activation of phospholipase
A2 followed by formation of the
arachidonic acid and its derivatives, which have been shown to inhibit
Na+-K+-ATPase
activity in rat nephron (30). However, the fact that Na+-K+-ATPase
was directly affected by TPA in isolated ciliary cell membranes (see
Fig. 2) reduces the possibility of involvement of signal transduction
pathways mediated by cytosolic systems.
For evaluation of the relative contribution of different
Na+-K+-ATPase
isoforms to the inhibitory effect of TPA on enzymatic activity, we
first quantitated the isoform composition in ciliary cell membranes
using a kinetic approach based on different ouabain sensitivities (7,
25). This approach revealed a two-component ouabain inhibitory response
of
Na+-K+-ATPase
that provides evidence for the existence of at least two enzyme
isoforms. Their IC50 values (2.6 and 409 nM) are in accordance with data previously reported for rat
3- and
2-isoforms of the Na+-K+-ATPase
-subunit, respectively (25, 28). Surprisingly, we did not observe
the typical low-affinity component (corresponding to the
1-subunit) in the micromolar
range of the ouabain concentration curve, since total inhibition of
enzyme activity already occurred at ~10 µM cardiac glycoside (see
Fig. 7A).
The most significant finding of our study is that only the fraction of
Na+-K+-ATPase
activity inhibitable by a low (50 nM) ouabain concentration was
susceptible to the inhibitory effect of TPA (Fig.
7B). This finding suggests that in
frog mucociliary membranes, PKC is capable of regulating only that
isoform that possesses a very high affinity for ouabain. Isoforms of
Na+-K+-ATPase
have been shown to exhibit different regulatory properties such as
kinetics of Na+ and
K+ activation (25) and other
features (see Ref. 31 for review). Several studies have assessed a
short-term regulation of different isoforms by hormones and
pharmacological agents (7, 14). For example, only the high-affinity
component of the rat synaptosomal Na+-K+-ATPase
(2) was found to be sensitive
to insulin treatment (7). Also, Kim et al. (20) have shown that
congestive heart failure or chronic norepinephrine infusion results in
a selective reduction of the cardiac
Na+-K+-ATPase
3-isoform.
We have previously shown that TPA enhancement of ciliary beat frequency is closely associated with intracellular Ca2+ accumulation independent of voltage-gated Ca2+ channels (21). Furthermore, in preliminary experiments, we found that the Na+-K+-ATPase inhibitor ouabain can also increase ciliary beat frequency and intracellular Ca2+ (unpublished data). These data suggest that PKC-mediated inhibition of Na+-K+-ATPase activity may play a role in regulation of ciliary beating by elevating intracellular Ca2+ levels via Na+/Ca2+ exchange.
In conclusion, we show that activation of PKC in intact frog ciliary cells results in specific inhibition of Na+-K+-ATPase activity. Moreover, the same effect was obtained by directly adding PKC activators to isolated cell membranes. The partial K+-p-NPPase activity of the enzyme was inhibited as well, but only in the presence of ATP, indicating that the nucleotide is ultimately required and that not only overall ATPase activity, but also its partial reaction, is subject to PKC regulation. Both high- and low-ouabain-affinity Na+-K+-ATPase forms were observed in this preparation, but only the high-affinity site was affected by TPA.
Further investigation will be needed to identify the molecular isoforms
of the
Na+-K+-ATPase
-subunit in frog esophagus mucociliary cell membranes and to
elucidate whether or not PKC-mediated inhibition of the enzyme activity
is associated with phosphorylation of the specific
-subunit.
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ACKNOWLEDGEMENTS |
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This work was supported by a grant from the Israel Science Foundation (to Z. Priel).
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FOOTNOTES |
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Address for reprint requests: M. Danilenko, Dept. of Clinical Biochemistry, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel.
Received 15 May 1997; accepted in final form 29 July 1997.
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