Several indirect lines of evidence suggest that protein kinases and phosphatases modulate the activity of renal Na+-K+-ATPase. The aim of this study was to examine whether such regulation may occur via modulation of the state of phosphorylation of Na+-K+-ATPase. Slices from rat renal cortex were prelabeled with [32P]orthophosphate and incubated with the inhibitors of protein phosphatase (PP)-1 and PP-2A, okadaic acid (OA) and calyculin A (CL-A), respectively, the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), or the PP-2B inhibitor, FK-506. Phosphorylation of Na+-K+-ATPase α-subunit was evaluated by measuring the amount of [32P]phosphate incorporation into the immunoprecipitated protein. Incubation with either OA, CL-A, or PDBu caused four- to fivefold increases in the amount of [32P]phosphate incorporation into immunoprecipitated Na+-K+-ATPase α-subunit. OA and PDBu had a synergistic effect on the state of phosphorylation of Na+-K+-ATPase α-subunit. FK-506 did not affect Na+-K+-ATPase phosphorylation, neither alone nor in the presence of PDBu. Each of the drugs, OA, CL-A, and PDBu, inhibited the activity of Na+-K+-ATPase in microdissected proximal tubules. PDBu potentiated OA-induced inhibition of Na+-K+-ATPase activity. Inhibition of Na+-K+-ATPase required a lower dose of CL-A than of OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. The PP-1 activity in rat renal cortex was ∼1.5 nmol Pi ⋅ mg protein−1 ⋅ min−1. Using a monoclonal anti-α antibody that fails to recognize the subunit when Ser23 is phosphorylated by PKC, we demonstrated that the dose response of PDBu inhibition of Na+-K+-ATPase correlated with the dose response of phosphorylation of the enzyme. The results suggest that the state of phosphorylation and activity of proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases.
- renal cortical tissue
- proximal convoluted tubule
- phorbol 12,13-dibutyrate
renalNa+-K+-ATPase plays a pivotal role for tubular Na+ reabsorption by generating the electrochemical gradient necessary for transcellular Na+ transport. It is well established that the activity of renal tubular Na+-K+-ATPase is bidirectionally regulated by natriuretic and antinatriuretic hormones, which, via G protein-coupled receptors, act on a common intracellular signaling pathway (2). Several indirect lines of evidence suggest that activation/deactivation of Na+-K+-ATPase by reversible phosphorylation is the final step in this pathway (4, 6,25).
To test this hypothesis, we have examined whether, in intact renal tissue, the phosphorylation state of Na+-K+-ATPase can be affected by various inhibitors of protein phosphatases such as okadaic acid (OA) and calyculin A (CL-A), which inhibit protein phosphatase (PP)-1 and PP-2A (14, 23), and FK-506, which inhibits protein phosphatase-2B (PP-2B) (26), or by an activator of protein kinase C (PKC), phorbol 12,13-dibutyrate (PDBu). The studies were performed on slices from rat outer renal cortex, a tissue that mainly consists of proximal tubular cells. The effect of OA on PP-1 activity in renal cortex was also evaluated. In separate protocols, we determined the effects of OA, CL-A, or PDBu on Na+-K+-ATPase activity in microdissected renal proximal tubules. The results support the concept that in intact renal tissue, the activity of Na+-K+-ATPase is dynamically modulated by phosphorylation/dephosphorylation reactions.
MATERIALS AND METHODS
Chemicals. OA was purchased from Scientific Marketing (Barnet Herts, UK); CL-A and BSA were from Boehringer (Mannheim, Germany); FK-506 was from Fujisawa Pharmaceutical (Osaka, Japan); DMSO was from LabKemi (Stockholm, Sweden); PDBu, EDTA, EGTA, NaF, phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, antipain, pepstatin A, chymostatin, β-mercaptoethanol, SDS, collagenase, and disodium ATP grade II were from Sigma (St. Louis, MO); ouabain was from Merck (Darmstadt, Germany); [32P]orthophosphoric acid (specific activity 8,500–9,120 Ci/mmol) and [γ-32P]ATP (specific activity 10 Ci/mmol) were from New England Nuclear (Boston, MA); Protein A Sepharose CL-4B was from Pharmacia (Uppsala, Sweden); rabbit affinity-purified antibody to mouse IgG was from Cappel (Durham, NC); and Malachite Green phosphatase assay kit was from Upstate Biotechnology (Lake Placid, NY).
Antibody. Mouse monoclonal antibody (MAb) 6H raised against α1-subunit of Na+-K+-ATPase was used for immunoprecipitation. It was produced using microsomal preparations of outer renal medulla of dog and rat enriched for Na+-K+-ATPase (30). As shown in Fig.1 A, phosphorylation of Na+-K+-ATPase does not affect the ability of the antibody to immunoprecipitate the protein. Accordingly, immunoblots obtained from samples treated with or without PDBu yielded equivalent α1-subunit immunoreactive signal.
32P-labeling and drug incubation of renal outer cortical slices.
Male Sprague-Dawley rats (39–45 days, 150–200 g body wt) were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). The animals were bled by cutting the abdominal aorta, and the kidneys were removed and decapsulated. A 200-μm-thick slice was taken from the superficial cortex using a Stadie-Riggs microtome. Each slice was preincubated at 30°C for 15 min in 2 ml of low-phosphate Krebs bicarbonate buffer (in mM: 124 NaCl, 4 KCl, 26 NaHCO3, 1.5 CaCl2, 1.5 MgSO4, 0.25 KH2PO4, and 10 d-glucose), bubbled with 95% O2-5% CO2. The slice was then incubated with 2.5 mCi of [32P]orthophosphoric acid in 2 ml of the same buffer at 30°C for 60 min to radiolabel the intracellular ATP pool. At the end of the labeling, the buffer was removed, and the 32P-labeled renal cortical slice was rinsed twice with 2 ml of fresh buffer. Slices from each sample were then incubated for the time indicated in the legends to Figs. 2 and 3 with drugs or DMSO (final concentration was less than 0.5%). The reaction was terminated by removing the buffer and rapidly freezing the tissues in dry ice and ethanol. The samples were stored at −70°C.
Immunoprecipitation of Na+-K+-ATPase.
Each slice was sonicated in 1 ml of cold lysis buffer (20 mM Tris hydrochloride, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 0.2% BSA; pH 8.0) containing 50 mM NaF, 1 mM EGTA, 25 mM benzamidine, 0.1 mM PMSF, 20 μg/ml leupeptin, 20 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml chymostatin. Aliquots from each sample containing equal amounts of protein were used for immunoprecipitation. Lysates were subsequently precleared at 4°C for 30 min with 10 mg of preswollen protein A-Sepharose CL-4B (final concentration of protein A-Sepharose was 1%) to get rid of nonspecific IgG and labeled proteins that bind nonspecifically to the beads. The beads were spun down for 15 s at 10,000g. The supernatants were incubated at 4°C for 1.5 h on a rotatory shaker with 15 μl of MAb 6H antibody (final dilution ∼1:100), followed by incubation with 25 μl of affinity-purified rabbit anti-mouse antibody (final dilution ∼1:80) at 4°C for 1 h. The resultant immunocomplexes were incubated with protein A-Sepharose beads at 4°C for 1 h. The beads were collected by centrifugation and washed at 4°C first with 1 ml of lysis buffer, then three times with 1 ml of a buffer containing 20 mM Tris ⋅ HCl, 150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.1% SDS, and 0.2% BSA (pH 8.0); three times with 1 ml of a buffer containing 20 mM Tris ⋅ HCl, 500 mM NaCl, 0.5% Triton X-100, and 0.2% BSA (pH 8.0); and finally with 1 ml of a buffer containing 50 mM Tris ⋅ HCl (pH 8.0). After the final wash, the beads were resuspended in 50 μl of 2× Laemmli sample buffer, vortexed, and centrifuged. The supernatants were resolved by electrophoresis on 7.5% SDS-polyacrylamide gel (24). Gels were dried and subjected to autoradiography.32P-labeled immunoprecipitated Na+-K+-ATPase α1-subunit was identified by its ability to migrate to the same position on SDS-PAGE as radiolabeled purified renal Na+-K+-ATPase α1-subunit (Fig.1 B). [32P]phosphate incorporation into the catalytic α-subunit of Na+-K+-ATPase was quantified with a LKB UltroScan XL Laser Densitometer interfaced to an IBM PC. Uneven background from lane to lane was corrected by subtraction. Results were expressed as a ratio of integrated absorbance units (IAU) of treated group vs. IAU of control group.
Immunoblotting and determination of phosphorylation. Renal outer cortical slices (200 μm thick) were incubated for 20 min in DMEM with or without addition of PDBu. Incubation was stopped by transferring slices into Laemmli sample buffer containing 50 mM NaF, 1 mM EGTA, 25 mM benzamidine, 0.1 mM PMSF, 20 μg/ml leupeptin, 20 μg/ml antipain, 5 μg/ml pepstatin A, and 5 μg/ml chymostatin. Samples were homogenized and centrifuged briefly. Ten micrograms of total proteins from each sample were loaded onto 7.5% SDS-polyacrylamide gel. Phosphorylation of Na+-K+-ATPase α1-subunit was assessed with Mck1, an antibody that detected specifically the PKC dephosphorylated form, but not phosphorylated form, of rat Na+-K+-ATPase (18). The antibody was a kind gift from Dr. Kathleen J. Sweadner (Laboratory of Membrane Biology, Massachusetts General Hospital). Immunoblotting and determination of Na+-K+-ATPase α1-subunit phosphorylation were performed as described (12). Results were expressed as a ratio of IAU of treated group vs. IAU of control group.
Determination of calcium/magnesium-independent, serine/threonine protein phosphatase (PP) activity. Renal outer cortical slices (200 μm thick) were homogenized in cold PP assay buffer composed of 50% glycerol, 20 mM MOPS (pH 7.5 at room temperature), 60 mM β-mercaptoethanol, 0.1 M NaCl, and 1 mg/ml BSA. The homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatants, termed “crude extracts,” were collected. PP activity in the crude extracts was measured with Malachite Green phosphatase assay kit, in which a serine/threonine phosphatase substrate, phosphopeptide (KRpTIRR), was used as the substrate. In preliminary experiments, crude extracts were checked for linearity of PP activity with respect to protein concentration and reaction time. Under these conditions, the amount of dephosphorylated substrate never exceeded 30% of the phosphorylated substrate present at the beginning of the reaction. About 1.5 mg/ml of protein from crude extracts was used in the assay, and the reaction time was 10 min. After incubation with vehicle or with 10 nM of OA at 4°C for 5 min, aliquots of 15 μl were transferred from each sample to a multi-well microtiter plate. The reaction was started by addition of phosphopeptide substrate (KRpTIRR) and carried out in a final volume of 25 μl at room temperature for 10 min in the presence or absence of phosphorylated Thiol-Cys34-d-32 peptide (40 nM), a potent and selective inhibitor of PP-1 (3, 22). This peptide contains the PP-1 binding site as well as the cAMP-dependent protein kinase (PKA) phosphorylation site of DARPP-32, a dopamine- and cAMP-dependent phosphoprotein of 32 kDa. DARPP-32 is, following phosphorylation by PKA, converted into a selective PP-1 inhibitor (21). The peptide was synthesized by solid-phase method using a model 430A Applied Biosystems peptide synthesizer and was phosphorylated by incubation with PKA. The characteristics of the phosphorylated DARPP-32 peptide has been described (22). The concentration of phosphorylated DARPP-32 peptide used is sufficient to block PP-1 (22). The reaction was then terminated by adding 100 μl of Malachite Green solution, and the samples were let to stand for 15 min at room temperature for color development. The absorbance of each well was measured in a microtiter plate reader at 650 nm. Absorbance from PP-1 was calculated as the difference between the absorbances from the total PP and the phosphorylated DARPP-32 peptide-insensitive PP. The amount of phosphate released was determined by comparing the absorbance to a standard curve prepared by incubating a set of phosphate standards of known concentration with Malachite Green solution. PP-1 activity was expressed as nanomoles of Pireleased per milligram protein per minute. Results are given as percent of control.
Preparation of tubules. Kidney perfusion and tubular microdissection were performed as described (15). Briefly, after a midline incision, the left kidney was exposed and perfused with 10 ml of cold Ringer solution and then with 40 ml of cold collagenase solution containing (in mM) 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 1 CaCl2, 1 MgCl2, and 10 Tris ⋅ HCl, as well as 0.05% collagenase and 0.1% BSA, pH 7.4, at 4°C. Kidney blood flow was not interrupted before the perfusion. The kidney was removed and cut along the corticopapillary axis into small pyramids, which were incubated at 35°C for 20 min in 10 ml of the same collagenase solution bubbled with 95% O2-5% CO2. After incubation, the pyramids were rinsed three times with cold microdissection solution, which was identical to the collagenase solution except that collagenase and BSA were omitted and CaCl2concentration was lowered to 0.25 mM.
The proximal convoluted tubule (PCT) segments were manually dissected from the superficial cortex at 4°C with the help of a stereomicroscope. They were individually transferred to the concavity of a bacteriological slide and photographed for length determination in an inverted microscope at ×100 magnification. The slides were stored on ice until assay.
Preincubation of tubules with drugs.The tubule segments were preincubated at room temperature for 20 min in 1 μl of microdissection solution with the addition of CL-A, OA, and/or PDBu, or vehicle and were transferred to ice. The segments were permeabilized with hypotonic shock, rapid freezing, and thawing to ensure that Na+ and ATP entered the cell. The Na+concentration in the medium ([Na+]m) was 70 mM. Permeabilization equalizes the intracellular Na+ concentration ([Na+]i) with the [Na+]m, thereby eliminating the transmembrane Na+ gradient and the possibility that changes in Na+-K+-ATPase activity are secondary to changes in [Na+]i.
Determination of Na+-K+-ATPase activity.
Na+-K+-ATPase activity was measured as described (15). All Na+-K+-ATPase assays were performed in the presence of saturating concentrations of all major substrates (70 mM Na+, 5 mM K+, and 10 mM ATP). After preincubation and permeabilization, the tubule segments were incubated at 37°C for 15 min in the following solution (in mM): 50 NaCl, 5 KCl, 10 MgCl2, 1 EGTA, 100 Tris ⋅ HCl, 10 disodium ATP, and 2–5 Ci/mmol [γ-32P]ATP in tracer amount (5 nCi/ml), pH 7.4, at 37°C, with or without 2 mM ouabain. When ouabain was present, NaCl and KCl were omitted, and Tris ⋅ HCl was 150 mM. The phosphate liberated by hydrolysis of [γ-32P]ATP was separated by filtration through a Millipore filter after absorption of the unhydrolyzed ATP on activated charcoal. The radioactivity was measured in a liquid scintillation spectrophotometer. Total ATPase and ouabain-insensitive ATPase activity were measured in separate samples, each consisting of five to eight segments. Na+-K+-ATPase activity was calculated as the difference between total ATPase and ouabain-insensitive ATPase activity and was expressed as picomoles of [32P]phosphate hydrolyzed per millimeter tubule per hour. Results are given as percent of control.
Statistical analysis. Values are means ± SE. Data were analyzed by Student’st-test, ANOVA test, and two-way ANOVA test. P < 0.05 was considered significant.
Incubation of renal slices with an inhibitor of PP-1 and PP-2A, OA (5 μM) or CL-A (5 μM), for 30 min, increased Na+-K+-ATPase α1-subunit phosphorylation by 5.4- and 5.5-fold, respectively (Fig. 2,A andB). The time course of the effect of OA is shown in Fig. 2 C. The increase in the amount of [32P]phosphate incorporation into Na+-K+-ATPase α1-subunit was maximal after 30 min of incubation and still present after 60 min.
Incubation with an activator of PKC, PDBu (5 μM), for 20 min, resulted in a 5.5-fold increase in the amount of [32P]phosphate incorporation into Na+-K+-ATPase α1-subunit (Fig.3, A andC). In slices incubated for 20 min with PDBu (5 μM) and OA (5 μM), a synergistic, 29-fold increase in the state of phosphorylation of Na+-K+-ATPase α1-subunit was observed (Fig. 3,A andC). The specific inhibitor of the calcium/calmodulin-dependent PP-2B, FK-506 (26), did not increase the state of phosphorylation of Na+-K+-ATPase α1-subunit and did not enhance the phosphorylation induced by PDBu (Fig. 3,B andC).
To evaluate the effect of OA on PP-1 activity, crude extracts from renal outer cortical slices were incubated with vehicle alone or with 10 nM OA. This method does not distinguish very well between PP-1 and PP-2A activity. To get an approximate measurement of PP-1 activity, we added to the assay the specific endogenous PP-1 inhibitor, DARPP-32, which in its phosphorylated state inhibits PP-1 (21). In this assay a synthetically produced phosphorylated DARPP-32 peptide was used. Using this approach, we found that OA inhibited PP-1 activity by 25.4 ± 3.9% (P < 0.05 vs. control by Student’s t-test). The contribution of PP-1 to total serine/threonine PP activity in renal cortex was ∼30%.
To determine the functional consequences of phosphorylation, Na+-K+-ATPase activity was measured in microdissected PCT in the presence or absence of OA, CL-A, or PDBu. All three substances inhibited PCT Na+-K+-ATPase activity in a concentration-dependent manner. OA and CL-A caused maximal inhibition at the concentration of 1 μM and 10 nM, respectively. The apparent half-maximal inhibitory concentration was ∼3.6 nM for OA and ∼0.14 nM for CL-A. The threshold concentration for inhibition was 1 nM for OA and 0.1 nM for CL-A (Fig.4). PDBu inhibited PCT Na+-K+-ATPase activity maximally at the concentration of 5 μM. The threshold concentration was 0.1 μM (Fig.5 A). A subthreshold concentration (10 nM) of PDBu significantly enhanced the inhibitory effect of OA (Fig. 6).
To compare the dose responses between inhibition of Na+-K+-ATPase activity and phosphorylation of the α-subunit, we studied PKC-induced phosphorylation using a PKC site-selective dephosphorylation-specific MAb developed by Feschenko and Sweadner (18). One advantage of this method is that it is not necessary to deprive the renal tissue of phosphate. The method also minimizes the risks for protein degradation and dephosphorylation. In addition, it identifies the specific phosphorylation site. As shown in Fig.5 B, PDBu triggered dose-dependent phosphorylation of the Na+-K+-ATPase α1-subunit. The dose response of PDBu inhibition of Na+-K+-ATPase activity correlated with the dose response of phosphorylation of the α-subunit (Fig. 5, A andB).
It is well documented that Na+-K+-ATPase purified from rat renal cortex can be phosphorylated in vitro by PKC (7, 17, 27). Phosphorylation occurs on Ser23 of the α1-subunit and is accompanied by a shift in the equilibrium between the E1 and the E2 forms of the enzyme (27). It is shown here that activation of PKC leads to phosphorylation of Na+-K+-ATPase α1-subunit in intact rat renal tissue as well as inhibition of the enzyme activity.
Previous studies on the effect of PKC activation on the function of Na+-K+-ATPase in intact cells have given controversial results. In an opossum kidney cell line (29) and in dissected rat renal proximal tubular segments and in rat choroid plexus (19), activation of PKC by phorbol esters was found to cause inhibition of Na+-K+-ATPase. Opposite effects, i.e., stimulation of Na+-K+-ATPase activity, were observed when rat renal proximal tubules in suspension (10) and hepatocytes (28) were incubated with phorbol esters. These controversial results may depend on species and tissue differences as well as on different experimental conditions. One such condition is the level of [Ca2+]i. A stimulatory effect is often observed at experimental conditions where the [Ca2+]iis high (28, 20) or might have been rendered high by manipulations such as using a K+-free solution (31) or hypertonic medium (400 mosmol/kgH2O) (10). K+-free pretreatment will inhibit the activity of Na+-K+-ATPase and then increase [Na+]i. The latter may increase [Ca2+]ivia inhibition of the Na+/Ca2+exchanger (9). Hypertonic medium will result in a decrease in cell volume, which triggers an increase in [Ca2+]i(16, 32). We have found in ongoing studies that in COS cells expressing rat renal Na+-K+-ATPase, activation of PKA or PKC caused inhibition of Na+-K+-ATPase at low [Ca2+]i(125 nM) and stimulation or no change at high [Ca2+]i(450 nM) (11).
In the present study, it was also shown that inhibition of PP-1 and PP-2A by OA and CL-A increased the state of phosphorylation of the Na+-K+-ATPase α1-subunit and inhibited the activity of Na+-K+-ATPase in rat kidney. OA and CL-A are known to inhibit PP-2A with the same potency, but CL-A is a more efficient inhibitor of PP-1 than OA (14,23). Our concentration-dependence curves indicated that CL-A inhibited Na+-K+-ATPase activity more efficiently than OA. This suggests that PP-1 rather than PP-2A is involved in the regulation of Na+-K+-ATPase. The PP-1 activity in rat renal cortex was found to be ∼30% of total calcium/magnesium-independent, serine/threonine protein phosphatase activity.
A large number of first messengers acting on renal tubular cells use PKC as an intracellular messenger. Thus dopamine regulation of renal tubular Na+-K+-ATPase has been partially attributed to PKC activation (1, 8). We have found in a preliminary study that the dopamine precursor,l-dopa, caused a ∼2-fold increase in the phosphorylation of Na+-K+-ATPase α1-subunit (data not shown).
Since PKC phosphorylation sites are generally good substrates for PP-1 and PP-2A (13), the effects of OA and CL-A on Na+-K+-ATPase are likely to be mediated via blockade of dephosphorylation on Ser23 of the α1-subunit, i.e., the PKC site. This hypothesis was supported by the observation that PDBu and OA had a synergistic effect on Na+-K+-ATPase phosphorylation. A synergistic inhibition of Na+-K+-ATPase activity was also observed when renal tubules were incubated with a subthreshold concentration of PDBu and different concentrations of OA.
The fact that phosphorylation and inhibition by PDBu occurred in the same dose range supports the concept that there is a direct link between the state of phosphorylation and the level of activity of the enzyme. In previous studies from this laboratory, it was shown that mutation of PKC phosphorylation site abolishes the increase in [Na+]iresponding to PDBu-induced inhibition of ion transport activity of Na+-K+-ATPase (5), and mutation of PKA phosphorylation site blocks the inhibition of Na+-K+-ATPase activity caused by cAMP agonists (12); also, a study on purified Na+-K+-ATPase showed that phosphorylation of Na+-K+-ATPase by PKC occurs on Ser23 of the α1-subunit and is accompanied by a shift in the equilibrium between the E1 and the E2 forms of the enzyme, thereby facilitating inhibition of Na+-K+-ATPase (27). Taken together, these observations indicate that phosphorylation of the enzyme may change its function and transporting capacity. It can, however, not be ruled out that the activity of the enzyme is also modulated by the state of phosphorylation of an intermediate protein.
In conclusion, the results presented in this study suggest that the state of phosphorylation and activity of renal proximal tubular Na+-K+-ATPase are determined by the balance between the activities of protein kinases and phosphatases. PKC and PP-1 appear to play an important role for this balance.
This study was supported by grants from the Swedish Medical Research Council (Project no. 03644), the Swedish Heart Lung Foundation and the Foundation of Axel Tielman’s Memory (to A. Aperia), and by Swedish Medical Research Council B96–14X-11580–01A (to G. Fisone).
Address for reprint requests: A. Aperia, Dept. of Woman and Child Health, Pediatric Unit, Astrid Lindgren Children’s Hospital, Karolinska Hospital, S-171 76 Stockholm, Sweden.
- Copyright © 1998 the American Physiological Society