Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

Irena Gertsberg, Irena Brodsky, Zvi Priel, and Michael Danilenko

Departments of Chemistry and Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(beta -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 4alpha -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).

Phorbol esters, diC8, and staurosporine were dissolved in a minimal volume of dimethyl sulfoxide (DMSO). Stock solution of alamethicin was prepared in 60% ethanol. The final concentrations of DMSO or ethanol in assay buffers never exceeded 0.1%.

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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of cell treatment with protein kinase C (PKC) activators on p-nitrophenylphosphatase (p-NPPase) activity. Frog esophagi were incubated with or without 80 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) or 20 µg/ml 1,2-dioctanoyl-sn-glycerol (diC8) in presence or absence of 100 nM staurosporine (SSP) as described in MATERIALS AND METHODS. K+-dependent (open bars) and -independent (hatched bars) p-NPPase activities were then determined in ciliary cell membranes in absence of test agents. Data are expressed as percentage of control activities measured in membranes from nontreated tissue. Control K+-dependent and -independent p-NPPase activities were 0.63 ± 0.05 and 1.07 ± 0.03 µmol p-nitrophenol · mg protein-1 · h-1, respectively. Data are means ± SE for 4 or 5 experiments. * P < 0.05.

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 4alpha -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).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of TPA (A) and diC8 (B) on ATPase activity in membranes. Na+-K+-ATPase (open circle ) and ouabain-insensitive ATPase (bullet ) activities were determined in presence or absence of TPA (0-1,000 nM) or diC8 (0-20 µg/ml) in ciliary cell membranes from nontreated esophagi. Data are expressed as percentage of control activities measured in absence of test agents. Control Na+-K+-ATPase and ouabain-insensitive ATPase activities were 4.30 ± 0.15 and 5.63 ± 0.18 µmol ADP · mg protein-1 · h-1, respectively. Data are means ± SE for 3 or 4 experiments. * P < 0.05. ** P < 0.01.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Time course of inhibitory effect of TPA on Na+-K+-ATPase activity in membranes. ATP hydrolysis was measured as rate of NADH oxidation at 340 nm in absence (open circle ) and presence (bullet ) of 10 nM TPA. Rates of ouabain-sensitive NADH oxidation from a representative experiment are shown. Similar results were obtained in at least 10 independent experiments. OD, optical density.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibitory effects of TPA and diC8 on Na+-K+-ATPase activity are mediated by PKC activation. A: Na+-K+-ATPase activity was determined in membranes in presence of TPA (open bars) or 4alpha -phorbol (hatched bars). B: Na+-K+-ATPase activity was measured in presence of 10 nM TPA, 20 µg/ml DiC8, with or without 100 nM staurosporine (SSP). Results were normalized to control (without additions). Control Na+-K+-ATPase activity was 3.62 ± 0.25 µmol ADP · mg protein-1 · h-1. Data are means ± SE for 4 or 5 experiments. * P < 0.05.

In contrast to their effects in whole cells (see Fig. 1), the PKC activators failed to affect K+-p-NPPase activity in direct membrane assays performed in the absence of ATP (Fig. 5A). However, in the presence of 100 µM ATP, the inhibitory effects were restored (Fig. 5B) to a level similar to that observed in the Na+-K+-ATPase assays (see Figs. 2 and 4). Such ATP dependence supports the involvement of protein kinase in the TPA inhibitory effect. In addition, it demonstrates that sensitivity to PKC activation is not limited to the overall ATPase activity of Na+-K+-ATPase, but is also a characteristic of its partial phosphatase reaction.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   ATP-dependent inhibition of K+-p-NPPase activity in membranes by TPA (A) and diC8 (B). K+-dependent p-NPPase activity was determined in absence (open bars) or presence (hatched bars) of 0.1 mM ATP as described in MATERIALS AND METHODS. Data are expressed as percentage of control (without additions). Control K+-p-NPPase activities with and without ATP were 0.36 ± 0.04 and 0.47 ± 0.08 µmol p-nitrophenol · mg protein-1 · h-1, respectively. Data are means ± SE for 4 or 5 experiments. * P < 0.05.

Because different isozymes of PKC are known to exhibit either Ca2+-dependent (e.g., alpha , beta , and gamma ) or -independent (e.g., delta , eta , or µ) activities (26), we determined whether the TPA action on Na+-K+-ATPase is influenced by Ca2+. As shown in Fig. 6A, Ca2+ alone caused a concentration-dependent decrease in Na+-K+-ATPase activity (IC50 = 2.6 ± 0.4 µM). However, the inhibitory effect of 10 nM TPA measured in the presence of two different concentrations of free Ca2+ (1 and 10 µM) was additive to that produced by Ca2+ itself and did not differ relatively from the effect exhibited in the absence of Ca2+ (Fig. 6B). These data suggest that the PKC isozyme(s) that mediates TPA-induced inhibition of Na+-K+-ATPase activity in frog mucociliary cell membrane belongs to the Ca2+-independent family.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   Inhibition of Na+-K+-ATPase by TPA is Ca2+ independent. A: dose-dependent inhibition of Na+-K+-ATPase activity by Ca2+. B: additive effects of Ca2+ and TPA. Na+-K+-ATPase activity was measured in absence (open bars) or presence (hatched bars) of TPA. Data are expressed as percentage of control (without additions). Control Na+-K+-ATPase activity in A and B was 4.24 ± 0.19 and 3.53 ± 0.19 µmol ADP · mg protein-1 · h-1, respectively. Data are means ± SE for 3 or 4 experiments. * P < 0.05. ** P < 0.01.

Three molecular isoforms of Na+-K+-ATPase have been characterized that differ in their sensitivity to cardiac glycosides as well as in other enzymatic properties and tissue distribution (31). To determine whether mucociliary cell membranes contain one or more Na+-K+-ATPase forms, we utilized a kinetic assay of enzyme inhibition by ouabain (7, 25). A complex pattern of ouabain concentration-response curve was observed (Fig. 7A). A shallow slope of the curve, which spans about five orders of magnitude, indicates the presence of multiple isoforms of the enzyme. Up to 30% of Na+-K+-ATPase activity was inhibited by 40 nM ouabain, and complete inhibition occurred with ~10 µM drug. Computer analysis of these data (see MATERIALS AND METHODS) revealed the best fit (r2 = 0.998) with a two-component inhibition (Fig. 7A). Calculations indicate that at least two enzyme forms with ouabain IC50 values of 2.6 ± 0.8 and 409 ± 65 nM are present in the mucociliary cell membranes with the contribution to total drug inhibition of 34.2 ± 2.3 and 62.6 ± 3.4%, respectively. It was, therefore, interesting to examine sensitivity to TPA of these two Na+-K+-ATPase forms. The form possessing a high affinity for ouabain was considered to be that inhibited by 50 nM drug (inflection point of the ouabain dose-dependence curve; see Fig. 7A), and the lower affinity form was determined as the difference between ATPase activities measured at 50 nM and 1 mM ouabain. Figure 7B demonstrates that the high-affinity Na+-K+-ATPase form was almost totally inhibited by 10 nM TPA with IC50 at ~1 nM, whereas its counterpart was only barely sensitive to the PKC activator. These results indicate that the regulatory action of PKC is Na+-K+-ATPase isoform specific.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7.   Na+-K+-ATPase isoforms of mucociliary cell membranes display different sensitivity to PKC activation. A: dose-dependent inhibition of Na+-K+-ATPase activity by ouabain. Control Na+-K+-ATPase activity was 4.13 ± 0.22 µmol ADP · mg protein-1 · h-1. Data are expressed as percentage of total Na+-K+-ATPase inhibition by 1 mM ouabain. Results were fitted to a two-component general logistic function as described in MATERIALS AND METHODS. Data are means for 6 experiments. Standard errors were 5-7%. B: differential effects of TPA on Na+-K+-ATPase isoforms. Activity of high-affinity isoform (open bars) was calculated as that inhibited by 50 nM ouabain. Activity of lower-affinity isoform (hatched bars) was calculated as difference between activities measured at 50 nM and 1 mM ouabain. Data are means ± SE for 3 experiments. * P < 0.05. ** P < 0.01.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 eta -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 alpha -subunit of Na+-K+-ATPase in purified enzyme preparations. Thr-15 and Ser-16 were detected to be the PKC phosphorylation sites on the alpha 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 alpha -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 alpha 2- and alpha 3-subunits from different species are also phosphorylated by PKC, although to a lower extent than alpha 1 (2). Amphibian (Bufo marinus and Xenopus) alpha 1-subunits can be phosphorylated by PKC both in vivo and in vitro (2, 8). Phosphorylation by PKC of the amphibian alpha 2- and alpha 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 alpha -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 alpha -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 alpha 3- and alpha 2-isoforms of the Na+-K+-ATPase alpha -subunit, respectively (25, 28). Surprisingly, we did not observe the typical low-affinity component (corresponding to the alpha 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 (alpha 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 alpha 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 alpha -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 alpha -subunit.

    ACKNOWLEDGEMENTS

This work was supported by a grant from the Israel Science Foundation (to Z. Priel).

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Beguin, P., A. T. Beggah, A. V. Chibalin, P. B. Burgenerkairuz, F. Jaisser, P. M. Mathews, B. C. Rossier, S. Cotecchia, and K. Geering. Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells: identification of a novel motif for PKC-mediated phosphorylation. J. Biol. Chem. 269: 24437-24445, 1994[Abstract/Free Full Text].

2.   Beguin, P., M. C. Peitsch, and K. Geering. alpha 1 but not alpha 2 or alpha 3 isoforms of Na,K-ATPase are efficiently phosphorylated in a novel protein kinase C motif. Biochemistry 35: 14098-14108, 1996[Medline].

3.   Bertorello, A. M., and A. Aperia. Na+-K+-ATPase is an effector protein for protein kinase C in renal proximal tubule cells. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25): F370-F373, 1989[Abstract/Free Full Text].

4.   Bertorello, A. M., A. Aperia, S. I. Walaas, A. C. Nairn, and P. Greengard. Phosphorylation of the catalytic subunit of the Na,K-ATPase inhibits the activity of the enzyme. Proc. Natl. Acad. Sci. USA 88: 11359-11362, 1991[Abstract].

5.   Bertorello, A. M., and A. I. Katz. Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F743-F755, 1993[Abstract/Free Full Text].

6.   Bradford, U. A rapid and sensitive method for the quantification of microgram quantities of proteins utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-255, 1976[Medline].

7.   Brodsky, J. L. Insulin activation of brain Na+-K+-ATPase is mediated by alpha 2-form of enzyme. Am. J. Physiol. 258 (Cell Physiol. 27): C812-C817, 1990[Abstract/Free Full Text].

8.   Chibalin, A. V., L. A. Vasilets, H. Hennekes, D. Pralong, and K. Geering. Phosphorylation of Na,K-ATPase alpha -subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C. J. Biol. Chem. 267: 22378-22384, 1992[Abstract/Free Full Text].

9.   Danilenko, M., P. Worland, B. Carlson, E. A. Sausville, and Y. Sharoni. Selective effects of mastoparan analogs: separation of G-protein-directed and membrane-perturbing activities. Biochem. Biophys. Res. Commun. 196: 1296-1302, 1993[Medline].

10.   Danilenko, M. P., V. C. Turmukhambetova, O. V. Yesirev, V. A. Tkachuk, and M. P. Panchenko. Na+-K+-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution. Am. J. Physiol. 261 (Cell Physiol. 30): C87-C91, 1991.

11.   Dong, J., N. A. Delamere, and M. Coca-Prados. Inhibition of Na+-K+-ATPase activates Na+-K+-2Cl- cotransporter activity in cultured ciliary epithelium. Am. J. Physiol. 266 (Cell Physiol. 35): C198-C205, 1994[Abstract/Free Full Text].

12.   Eshel, D., Y. Grossman, and Z. Priel. Spectral characterization of ciliary beating: variations in frequency with time. Am. J. Physiol. 249 (Cell Physiol. 18): C160-C165, 1985[Abstract/Free Full Text].

13.   Esmann, M. ATPase and phosphatase activity of Na+,K+-ATPase: molar and specific activity, protein determination. Methods Enzymol. 156: 105-115, 1988[Medline].

14.   Ewart, H. S., and A. Klip. Hormonal regulation of the Na+-K+-ATPase: mechanisms underlying rapid and sustained changes in pump activity. Am. J. Physiol. 269 (Cell Physiol. 38): C295-C311, 1995[Abstract/Free Full Text].

15.   Feschenko, M. S., and K. J. Sweadner. Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C. J. Biol. Chem. 269: 30436-30444, 1994[Abstract/Free Full Text].

16.   Feschenko, M. S., and K. J. Sweadner. Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C. J. Biol. Chem. 270: 14072-14077, 1995[Abstract/Free Full Text].

17.   Fisone, G., G. L. Snyder, J. Fryckstedt, M. J. Caplan, A. Aperia, and P. Greengard. Na+,K+-ATPase in the choroid plexus. Regulation by serotonin/protein kinase C pathway. J. Biol. Chem. 270: 2427-2430, 1995[Abstract/Free Full Text].

18.   Jean, T., and C. B. Klee. Calcium modulation of inositol 1,4,5-triphosphate-induced calcium release from neuroblastoma × glioma hybrid (NG 108-15) microsomes. J. Biol. Chem. 261: 16414-16420, 1986[Abstract/Free Full Text].

19.   Kawai, N., T. Yamamoto, H. Yamamoto, R. M. McCarron, and M. Spatz. Endothelin 1 stimulates Na+,K+-ATPase and Na+-K+-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium. J. Neurochem. 65: 1588-1596, 1995[Medline].

20.   Kim, C. H., T. H. Fan, P. F. Kelly, Y. Himura, J. M. Delehanty, C. L. Hang, and C. S. Liang. Isoform-specific regulation of myocardial Na,K-ATPase alpha -subunit in congestive heart failure. Role of norepinephrine. Circulation 89: 313-320, 1994[Abstract].

21.   Levin, R., A. Briaman, and Z. Priel. Protein kinase C induced calcium influx and sustained enhancement of ciliary beating by extracellular ATP. Cell Calcium 21: 103-113, 1997[Medline].

22.   Lynch, C. J., K. M. McCall, Y. C. Ng, and S. A. Hazen. Glucagon stimulation of hepatic Na+-pump activity and alpha-subunit phosphorylation in rat hepatocytes. Biochem. J. 313: 983-989, 1996[Medline].

23.   Middleton, J. P., W. A. Khan, G. Collinsworth, Y. A. Hannun, and R. M. Medford. Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells. J. Biol. Chem. 268: 15958-15964, 1993[Abstract/Free Full Text].

24.   Mito, T., and N. A. Delamere. Alteration of active Na-K transport on protein kinase C activation in cultured ciliary epithelium. Invest. Ophthalmol. Visual Sci. 34: 539-546, 1993.[Abstract]

25.   Munzer, J. S., S. E. Daly, E. A. Jewell-Motz, J. B. Lingrel, and R. Blostein. Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase. J. Biol. Chem. 269: 16668-16676, 1994[Abstract/Free Full Text].

26.   Nishizuka, Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484-496, 1995[Abstract/Free Full Text].

27.   Norby, J. G. Coupled assay of Na+,K+-ATPase activity. Methods Enzymol. 156: 116-119, 1988[Medline].

28.   O'Brien, W. J., J. B. Lingrel, and E. T. Wallick. Ouabain binding kinetics of the rat alpha 2 and alpha 3 isoforms of the sodium potassium adenosine triphosphate. Arch. Biochem. Biophys. 310: 32-39, 1994[Medline].

29.   Riley, M. V., and K. Kishida. ATPase of ciliary epithelium: cellular and subcellular distribution and probable role in secretion of aqueous humor. Exp. Eye Res. 42: 559-568, 1986[Medline].

30.   Satoh, T., H. T. Cohen, and A. I. Katz. Intracellular signaling in the regulation of renal Na-K-ATPase. II. Role of eicosanoids. J. Clin. Invest. 91: 409-415, 1993[Medline].

31.   Sweadner, K. J. Subunit diversity in the Na,K/ATPase. In: The Sodium Pump: Structure, Mechanism and Regulation, edited by J. H. Kaplan, and P. DeWeer. New York: Rockefeller Univ. Press, 1991, p. 63-76.

32.   Tarasiuk, A., M. Bar-Shimon, L. Gheber, A. Korngreen, Y. Grossman, and Z. Priel. Extracellular ATP induced hyperpolarization and motility stimulation of ciliary cells. Biophys. J. 68: 1163-1169, 1995[Abstract].


AJP Cell Physiol 273(6):C1842-C1848
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society