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RECEPTORS AND SIGNAL TRANSDUCTION

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity

Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts

Submitted 3 May 2005 ; accepted in final form 10 October 2005

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type.

protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal

In addition to signals transmitted from Na⁺-K⁺-ATPase to ERK1/2, signals can be transmitted in the opposite direction. Na⁺-K⁺-ATPase activity and transmembrane Na⁺-K⁺-ATPase current in renal cells was blocked by inhibition of ERK1/2 (22). Inhibition of ERK1/2 blocked the insulin-promoted stimulation of Na⁺-K⁺-ATPase and the phosphorylation of the -subunits of Na⁺-K⁺-ATPase in skeletal muscle and renal cells (2, 37). ERK1/2 also regulates the expression of the - and -subunits of Na⁺-K⁺-ATPase (9, 27).

Ouabain blocks net fluid and electrolyte secretion by epithelial cells, including secretion initiated by muscarinic receptor ligands in parotid and other salivary gland epithelial cells. The muscarinic receptor is a G protein-coupled receptor linked to PLC. Muscarinic stimulatory ligands sequentially increase the production of inositol 1,4,5-trisphosphate (InsP₃), increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i), and open Ca²⁺-sensitive ion channels and other ion transport proteins that initiate fluid secretion in the parotid gland. The resulting ionic alterations, including large increases in [Na⁺]_i and decreases in intracellular K⁺ concentration ([K⁺]_i), produce an increase in Na⁺-K⁺-ATPase activity (5, 25). By inhibiting Na⁺-K⁺-ATPase, ouabain blocks receptor-mediated increases in fluid secretion and saliva formation in salivary cells (20). Muscarinic receptor-initiated changes in ion movements are accompanied by changes in the phosphorylation and activity status of signaling proteins, including an increase in the activation of ERK1/2 (29).

In initial studies, in which we used freshly isolated parotid acinar cells, we investigated whether ouabain had a stimulatory effect on ERK1/2 activation in a manner similar to that found in many other cell systems. Although ouabain produced modest changes (generally less than a doubling in ERK1/2 phosphorylation), we found that ouabain greatly amplified the activation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol. In the present study, we examined this phenomenon in more detail and also examined changes in ERK1/2 phosphorylation by lowering extracellular K⁺ to block Na⁺-K⁺-ATPase. Because acute inhibition of ERK1/2 can affect the Na⁺-K⁺-ATPase activity of ex vivo cells and cells in culture (2, 22), we also examined whether ERK1/2 inhibition could alter Na⁺-K⁺-ATPase activity in freshly isolated rat parotid cells. Our studies suggest that within the same epithelial cell type, ouabain and low extracellular K⁺ promote distinct signals to initiate ERK1/2 activation and that ERK1/2 inhibition has a negative effect on Na⁺-K⁺-ATPase activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Chemicals. Carbamyl choline (carbachol) and N-acetyl-L-cysteine were purchased from Sigma (St. Louis, MO). Ouabain was purchased from Sigma-Aldrich and Calbiochem (San Diego, CA). 1,4-Diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), ATP (A-2383), and Fiske and Subbarow reducer were purchased from Sigma. 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF-109203X), 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) were purchased from Calbiochem. BAPTA-AM and fura-2 AM were purchased from Molecular Probes (Eugene, OR). Anti-phosphotyrosine antibody was a generous gift from Dr. Thomas M. Roberts (Dept. of Pathology, Dana Farber Cancer Institute, Boston, MA). PAb ERK2 (SC-154), MAb ERK2 (SC-1647), and MAb anti-₁-Na⁺-K⁺-ATPase (SC-21712) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-₁-Na⁺-K⁺-ATPase PAb (no. 05-369) was purchased from Upstate Biotechnology. PAb phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-p90 Rsk were purchased from Cell Signaling Technology (Beverly, MA). Secondary antibodies used with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) were IRDye 800-conjugated anti-rabbit IgG (no. 611-632-122; Rockland Immunochemicals, Gilbertsville, PA) and Alexa Fluor 680 anti-mouse IgG (A-21058; Invitrogen, Carlsbad, CA). Igepal was purchased from ICN Biomedicals (Irvine, CA). All other chemicals were of reagent grade or better.

Cell preparation and solutions. Animal care and the protocols followed for animals in all experiments were reviewed and approved by the Harvard Medical Area Standing Committee on Animals. Parotid acinar cells were prepared from male Sprague-Dawley rats (Charles River Laboratories, Kingston, NY, or Taconic, Germantown, NY; 175–225 g) according to previously established techniques (32). In brief, rat parotid glands were removed and treated with trypsin and collagenase to obtain a suspension of single cells and small groups of cells. Cells were suspended at 0.5–1 mg of protein/ml in solution A, which was composed of (in mM) 116.4 NaCl, 5.4 KCl, 1 NaH₂PO₄, 25 Na⁺-HEPES, 1.8 CaCl₂, 0.8 MgCl₂, 5 sodium butyrate, and 5.6 glucose, pH 7.4. K⁺-free solution A was made by replacing KCl with equimolar NaCl. Cells were maintained on ice before use.

All cell treatments were performed at 37°C in a water-jacketed chamber using a magnetic flea to stir the suspended cells. Aliquots of cells (usually 1.5 ml) were equilibrated for 2–3 min before treatment with various agents or vehicles (water or 0.1–0.2% DMSO). In general, cells were exposed to the Src family inhibitor PP2 (10 µM for 15 min), the PKC inhibitor GF-109203X (10 µM for 15 min), and the Ca²⁺ chelator BAPTA (25 µM for 20 min), or vehicle, followed by treatment with ouabain (1 mM) or vehicle (DMSO) for 1 min and stimuli for 2 additional min as indicated. In some experiments, cells were treated with various concentrations of ouabain before being treated with carbachol. Basal conditions represent cells treated with DMSO for 3 min. Cells exposed solely to ouabain were treated for 3 min. When experiments were conducted using K⁺-free solution, cells were preequilibrated at 37°C in normal solution A for 15 min, spun down, and resuspended in warmed (37°C) K⁺-free solution A for up to 10 min as indicated.

Western blot analysis. At the end of the treatment period, parotid cells were collected by rapid sedimentation in a microcentrifuge (model no. 5414; Brinkmann Instruments, Westbury, NY). The supernatant was removed, and cells were lysed in 400 µl of ice-cold lysis buffer [137 mM NaCl, 20 mM Tris base, pH 7.5, 1 mM EGTA, 1 mM EDTA, 10% glycerol (vol/vol), and 1% Igepal (vol/vol)] containing the following reagents: 1 mM vanadate, 4.5 mM sodium pyrophosphate, 47.6 mM NaF, 9.26 mM -glycerophosphate, 0.5 mM DTT, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 2 µg/ml aprotenin, and 2 µg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. The lysates were vortexed extensively and then sedimented at 15,000 g for 15 min at 4°C. The cleared supernatants were transferred to fresh microfuge tubes and diluted with 5x Laemmli sample buffer [312.5 mM Tris·HCl (pH 6.8), 25% glycerol, 5% SDS, 1.8 M -mercaptoethanol, and bromophenol blue (trace)], boiled for 5 min, and stored at –20°C before electrophoresis. Samples were separated using SDS-PAGE with a 10% separating gel and a 3% stacking gel as described previously (3). Proteins were transferred onto nitrocellulose membranes. Immunoblots were probed overnight with various antibodies according to the supplier's specifications. Proteins were visualized using ECL reagents and X-ray film; alternatively, in other experiments, proteins were visualized and quantified using direct infrared fluorescence with the Odyssey Infrared Imaging System.

Quantification of ERK1/2 activation. In all experiments, changes in the phosphorylation status of ERK1/2 were evaluated as an indication of changes in the activation of ERK. Unless indicated otherwise, changes in ERK1/2 activation and/or phosphorylation for various conditions were normalized to ERK1/2 phosphorylation under basal (nonstimulated) conditions in the absence of ouabain or other inhibitors. For each sample, phosphorylated ERK1/2 was normalized to total ERK2 to account for gel loading and/or transfer variations. Quantifications of ERK1/2 were performed in one of two ways. In some experiments, bands of relevant proteins on X-ray film (obtained using ECL techniques) were quantified using the National Institutes of Health's ImageJ software. Care was taken to use exposure times that were within the linear capability of the film. For these experiments, blots were probed for phosphoproteins, stripped, and reprobed for total proteins. In other experiments in which we used the Odyssey Infrared Imaging System, blots were probed simultaneously for phosphoproteins and total protein levels using PAbs and mouse MAbs, respectively, and fluorescent anti-rabbit and anti-mouse antibodies were used for visualization and subsequent quantification.

Measurement of [Ca²⁺]_i in parotid cells. Alterations in [Ca²⁺]_i were analyzed in parotid cells in suspension by measuring changes in the fluorescence of the Ca²⁺ indicator dye fura-2, similarly to the method described in previous studies (32). In brief, cells suspended in 2 µM fura-2 AM (in solution A plus 2% BSA) were incubated at 37°C for 60 min. Cell were washed, suspended in solution A, and maintained on ice. For experiments conducted in Ca²⁺-free media, aliquots of fura-2-loaded cells were incubated at 37°C in solution A for 5 min, rapidly washed twice in warmed (37°C) Ca²⁺-free solution A, resuspended in 2 ml of this solution, and added to the cuvette in a chamber maintained at 37°C. After 3 min, 100 µM EGTA was added to the solution, and 1 min later, the experiment was commenced. For experiments conducted in Ca²⁺-containing solution A, aliquots of cells were incubated at 37°C in solution A for 5 min, rapidly washed twice in solution A, resuspended in 2 ml of this solution, and added to the cuvette in a chamber maintained at 37°C. Fura-2 fluorescence was monitored at a 340/380-nm excitation ratio and 510-nm emission using a QuantaMaster fluorescence spectrophotometer (Photon Technology International, Birmingham, NJ). Generally, duplicates or triplicates of each condition were examined in each experiment, and the results were averaged. Changes in [Ca²⁺]_i were evaluated on the basis of changes in the 340/380-nm excitation ratio. Statistical analysis of the results was performed using data compiled from all independent experiments (n = 3).

Oxygen consumption. QO₂ measurements were similar to those reported previously (29). Parotid acinar cells suspended in solution A were prewarmed for 15 min at 37°C before being placed into a stirred 400-µl chamber maintained at 37°C. A Clark-type O₂ electrode (model 125/05; Instech Laboratories, Plymouth Meeting, PA) was used to measure the disappearance of O₂ from the closed chamber. Output was recorded using a flatbed chart recorder (model BD112; Kipp & Zonen, Bohemia, NY). At the end of each measurement, a sample of the cell suspension was collected for protein analysis (18) so that each QO₂ measurement could be normalized to the protein content of the cells. The QO₂ value was calculated as the linear QO_{2 max} upon addition of an agent to the chamber. The changes in QO₂ (QO₂) values for parotid cells were the differences between the sustained QO₂ under basal conditions and the linear QO_{2 max} upon addition of different concentrations of carbachol.

For experiments conducted using U0126, cells suspended in solution A were treated with 10 µM drug or 0.1% DMSO for 20 min at 37°C and then placed into the chamber containing the O₂ electrode. Basal QO₂ was monitored, and carbachol (10^–5 M) or nystatin (80 µg/ml) was added. As described above, the QO₂ values were calculated as the difference between the sustained basal QO₂ and linear QO_{2 max} upon the addition of carbachol and nystatin. In each of three experiments, five to nine individual samples were analyzed for each condition. In each experiment, the values from U0126-treated cells were normalized to the values from control cells.

ATP measurement. ATP was measured in parotid acinar cells as described previously (29). Cells suspended in solution A were treated for 20 min with vehicle or U0126 (10 µM) at 37°C. ATP was extracted from the cells and analyzed spectrophotometrically. This procedure was used in three independent experiments.

Na⁺-K⁺-ATPase activity assay. The Na⁺-K⁺-ATPase was measured on homogenates of parotid acinar cells. In brief, freshly isolated parotid acinar cells were washed once in a phosphate-free buffer and suspended in a homogenization buffer (in mM: 150 sucrose, 10 HEPES, pH 7.4, and 4 EGTA). The cells were homogenized using a glass Dounce homogenizer (20 up-and-down strokes), and the homogenate was frozen at –80°C. Aliquots of the homogenate were added to a Na⁺-K⁺-ATPase assay mixture (pH 7.4) consisting of (in mM) 140 NaCl, 20 KCl, 30 histidine, 0.2 EGTA, 3 MgCl₂, and different concentrations of ouabain. The samples were preincubated at 37°C for 5 min, and the reaction was started with the addition of 3 mM ATP. The reaction was stopped after 15 min by the addition of ice-cold TCA (3.2% final concentration). ATP hydrolysis (phosphate liberation) was measured using a modified Fiske-Subbarrow assay (8). Total Na⁺-K⁺-ATPase activity was defined as the ATPase activity that was sensitive to 3 mM ouabain, which was 48.6 ± 2.5% (n = 3) of the total ATPase activity. Each assay (n = 3) was conducted using a homogenate from a different preparation of acinar cells.

Data analysis. Values were calculated as means ± SE of the number (n) of independent experiments (each n representing a different cell preparation). Differences between control or basal and experimental samples for accumulated data were evaluated using a two-tailed Student's t-test. All experiments including Western blot analysis were performed at least three times. Within each experiment to be analyzed using Western blot analysis or the Odyssey Infrared Imaging System, multiple (i.e., duplicate or triplicate) cell samples were collected for each condition and subjected to SDS-PAGE, and the average of the values obtained within each individual experiment was treated as n = 1.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Ouabain potentiates the phosphorylation of ERK1/2 by carbachol. In preliminary experiments, ouabain did not produce substantial increases in ERK1/2 activation in rat parotid acinar cells exposed to ouabain (10^–6-10^–3 M) for up to 15 min (data not shown). Exposure of cells to 1 mM ouabain, a concentration sufficient to block the majority of the relatively ouabain-insensitive ₁-subunit of the rodent Na⁺-K⁺-ATPase, produced less than a doubling of basal ERK1/2 phosphorylation (Fig. 1, A and B, and data not shown). However, when 1 mM ouabain was added 1 min before a concentration of carbachol (10^–6 M) that produced only a small increase (50–100%) above basal levels in ERK1/2 activation when it was added by itself for 2 min, the combined presence of ouabain and carbachol activated ERK1/2 to a level similar to that produced by 10^–5 M carbachol (Fig. 1, A and B). Ouabain did not increase the activation of ERK1/2 by 10^–5 M carbachol (Fig. 1, A and B), which is a maximal concentration for ERK1/2 activation (Fig. 1C). Ouabain (1 mM) also potentiated ERK1/2 activation using 3 x 10^–7 M carbachol (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Ouabain (Ouab) potentiates ERK1/2 phosphorylation (P-ERK1/2) by submaximal concentrations of carbachol (Carb). A: cells were treated with 10–5 and 10–6 M carbachol for 2 min after a 1-min exposure to DMSO (–) or 1 mM ouabain. Other cells were exposed for 3 min to either ouabain or DMSO (–). Lysates were subjected to immunoblot (IB) analysis as indicated. B: quantification of ERK1/2 activation in cells treated with various concentrations of carbachol for 2 min after exposure to 1 mM ouabain for 1 min (Ouabain 1'). Some cells were exposed simultaneously to a combination of ouabain (1 mM) and carbachol (10–6 M) for 2 min (Ouabain 0'). Basal cells were exposed for 3 min to ouabain or DMSO. ERK1/2 activation was calculated relative to basal without ouabain. Note that ouabain and carbachol (10–6 M) added singly produced only modest increases in ERK1/2 activation. C: quantification of relative increases in ERK1/2 phosphorylation above basal level in cells exposed to different concentrations of carbachol for 2 min. Activation by 10–5 M carbachol was defined as maximal O2 consumption (QO2 max) (100) and was 7.2 ± 0.8 times (6) basal QO2 level. Numbers in parentheses are the number of independent experiments conducted.

Ouabain had a concentration-dependent effect in amplifying the activation of ERK1/2 by 10^–6 M carbachol (Fig. 2, A and B). The EC₅₀ value of ouabain in promoting this effect was 100 µM or higher (Fig. 2C). This finding is similar to the inhibitory potency of ouabain reported to block the Na⁺-K⁺-ATPase activity of rodent cells containing primarily the ₁-isoform of Na⁺-K⁺-ATPase (4, 6). Consistent with these data, we detected the ₁-subunit in parotid acinar cells using Western blot analysis with ₁-specific antibodies (data not shown). We also evaluated the potency of the effect of ouabain on the Na⁺-K⁺-ATPase enzymatic activity in parotid acinar cells using a biochemical assay to measure the ouabain-sensitive rate of ATP hydrolysis. The IC₅₀ of ouabain was 100 µM or higher (Fig. 2D), which was similar to its potency in amplifying the activation of ERK1/2 by 10^–6 M carbachol.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Concentration dependence of ouabain on ERK1/2 activation by 10–6 M carbachol and on inhibition of Na+-K+-ATPase activity. A–C: parotid acinar cells were treated for 1 min with different concentrations of ouabain before carbachol (2 min). A: representative Western blot experiment. B: quantification of ERK1/2 activation relative to basal level. Ouabain produced a concentration-dependent increase in ERK1/2 activation. C: increases in ERK1/2 activation above basal levels were normalized to the increase produced by the combination of ouabain (1 mM) + carbachol (10–6 M), which was defined as 100. Numbers in parentheses in B and C are the number of independent experiments conducted. D: Na+-K+-ATPase activity in homogenates of parotid acinar cells was measured in the presence of different concentrations of ouabain (n = 3 experiments).

View larger version (48K): [in this window] [in a new window]   Fig. 3. Contributions of various signaling proteins and events to ERK1/2 activation. Parotid acinar cells were treated with various inhibitors for 15–20 min and then exposed to ERK1/2-stimulating conditions. See EXPERIMENTAL PROCEDURES for details. A: concentration dependence of the Src family inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) in blocking ERK1/2 activation. Cells were exposed to 1 mM ouabain 1 min before 10–6 M carbachol for 2 min. B: PP2 (10 µM) blocked ERK1/2 activation by 10–5 M carbachol (2 min) and 100 nM PMA (2 min). C: concentration dependence of PKC inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF-109203X) in blocking ERK1/2 activation. Cells were exposed to 1 mM ouabain 1 min before 10–6 M carbachol (2 min). D: reduction of ERK1/2 activation in parotid cells loaded with the Ca2+ chelator BAPTA. Quantified data from multiple experiments for all of these conditions are presented in Table 1.

Removal of extracellular K⁺ stimulates ERK1/2 and p90 Rsk phosphorylation. The concentration dependence of ouabain on carbachol-initiated ERK1/2 activation (Fig. 2) suggests that its potentiating effects are due to inhibition of Na⁺-K⁺-ATPase. Because Na⁺-K⁺-ATPase activity is also inhibited by the removal of extracellular K⁺, we also examined the effects of low extracellular K⁺ on ERK1/2 activation (see EXPERIMENTAL PROCEDURES) and compared this with that of carbachol. ERK1/2 phosphorylation in cells suspended in K⁺-free solution increased within 2 min and reached a peak at 5 min that was sustained. The readdition of 5 mM K⁺ at 5 min resulted in a return of ERK1/2 phosphorylation to basal levels within 5 min (Fig. 4A). In contrast, the activation by carbachol (10^–5 M) peaked at 2 min and was not sustained at this level at later times. Both carbachol and K⁺-free conditions also increased the phosphorylation of p90 Rsk (Fig. 4B), an effector of ERK1/2. Moreover, the activation of ERK1/2 by K⁺-free conditions, like those of carbachol, were detected in cells exposed to the EGFR inhibitor AG-1478 at a concentration (300 nM) that effectively blocked the activation of ERK1/2 and p90 Rsk by EGF (Fig. 4B).

View larger version (32K): [in this window] [in a new window]   Fig. 4. Comparison of carbachol and K+-free (0K+) extracellular medium on ERK1/2 and p90Rsk phosphorylation in parotid acinar cells. A: cells were treated with 10–5 M carbachol or resuspended in K+-free solution for various times, and ERK1/2 phosphorylation was quantified relative to basal levels in normal K+-containing solution. Some cells were exposed to K+-free conditions for 5 min, followed by exposure to 5 mM K+ for an additional 5 min (n = 4–5 cell preparations). B: cells were pretreated with vehicle (–; DMSO) or 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478; 300 nM) for 15 min, followed by exposure to carbachol (10–5 M) for 2 min, K+-free solution for 5 or 10 min, and EGF (100 ng/ml) for 2 min. Lysates were immunoblotted for phospho-ERK1/2, ERK2, and phospho-p90Rsk. Each lane represents a different sample collected from the same cell preparation. Blots are representative of 3 experiments.

Ouabain does not enhance Ca²⁺ signaling by muscarinic receptors. Because exposure of the parotid cells to ouabain amplified the actions of a submaximal concentration of carbachol on ERK1/2, we wondered whether there would be an increase in the activation of M3 receptor itself. Also, because the activation of ERK1/2 by carbachol depended on an increase in [Ca²⁺]_i, we wondered whether ouabain would enhance the ability of 10^–6 M carbachol to increase the release of intracellular Ca²⁺. We examined this possibility by measuring the intracellular release of Ca²⁺ in carbachol-treated cells exposed to Ca²⁺-free solution. Added by itself, ouabain (1 mM) did not produce a detectable increase in [Ca²⁺]_i during the first minute of exposure (Fig. 5) or within the first 5 min of exposure (data not shown). In control cells (i.e., those treated with vehicle) and in cells exposed to 1 mM ouabain for 1 min before carbachol treatment, 10^–6 M carbachol caused the release of similar amounts of Ca²⁺ from intracellular storage sites, and the amounts of Ca²⁺ released were not as large as those promoted by 10^–5 M carbachol. The peak increases in [Ca²⁺]_i produced by 10^–6 M carbachol in the absence or presence of 1 mM ouabain were 46.5 ± 0.6% (n = 3) and 45.3 ± 1.73% (n = 3), respectively, of the increase produced by 10^–5 M carbachol. Within 2.5 min, [Ca²⁺]_i returned to basal levels after exposure to all types of stimulation. In experiments conducted using cells suspended in Ca²⁺-containing solution A, 10^–6 M carbachol produced similar changes in [Ca²⁺]_i in the presence and absence of ouabain (data not shown). Both the initial peak elevation and the sustained elevation in [Ca²⁺ ]_i were similar in cells with or without 1 mM ouabain present, and these responses also were much less than those produced by 10^–5 M carbachol.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effect of ouabain on the elevation of intracellular Ca2+ concentration ([Ca2+]i) by 10–6 M carbachol. [Ca2+]i was monitored in fura-2-loaded cells suspended in Ca2+-free solution. Cells were treated sequentially with EGTA (100 µM) immediately before start of recording, ouabain (1 mM), or DMSO, as well as two different concentrations of carbachol. Traces show averages of multiple trials conducted with one cell preparation. Ouabain did not increase the intracellular release of intracellular Ca2+ produced by 10–6 carbachol, and the release by 10–6 M carbachol ± ouabain was much less than that produced by 10–5 M carbachol. Ordinate axis is the 340/380-nm fura-2 fluorescence excitation ratio. See text for details.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Concentration dependence of carbachol on ERK1/2 activation and Na+-K+-ATPase activation. The ERK1/2 data are reproduced from Fig. 1C. The increases in the Na+-K+-ATPase activities were calculated as QO2 increases in parotid cells exposed to various concentrations of carbachol. See EXPERIMENTAL PROCEDURES for more details. In each QO2 experiment (n = 3), the QO2 above basal levels promoted by 10–5 M carbachol was defined as QO2 max (100), and other values shown are relative to this value. Basal QO2 = 11.6 ± 0.4 nmol O2·mg–1·min–1 (n = 3), and carbachol (10–5 M) QO2 = 9.6 ± 1.63 nmol O2·mg–1·min–1 (n =3) above basal level. For ERK, the activation produced by 10–5 M carbachol (n = 6) was 7.2 ± 0.8 times that of basal level.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effect of the MEK inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) on QO2 of rat parotid acinar cells. Cells were treated with vehicle (0.1% DMSO) or 10 µM U0126 for 20 min, after which either carbachol (10–5 M) or nystatin (80 µg/ml) was added. The effects of U0126 on basal QO2 and QO2 in response to carbachol and nystatin were calculated from 3–6 independent experiments as indicated. Values for U0126-treated cells were normalized to control values. The normalized carbachol and nystatin QO2 values do not include basal QO2 values. Control values (in nmol O2·mg–1·min–1) were as follows: basal QO2, 12.3 ± 0.5 (n = 6); carbachol QO2, 9.9 ± 1.9 (n = 3); nystatin QO2, 16.1 ± 2.3 (n = 4). Absolute differences in QO2 (in nmol O2·mg–1·min–1) between control and U0126-treated cells were as follows: basal, 0.8 ± 0.3 (n = 6); carbachol, 1.2 ± 0.3 (n = 3); nystatin, 1.9 ± 0.8 (n = 4). See EXPERIMENTAL PROCEDURES for details. *P = 0.6, **P < 0.01, ***P < 0.04 vs. control.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

The similarities between the ouabain concentration dependence of the potentiation of muscarinic ERK1/2 signaling (Fig. 2C) and the inhibition of Na⁺-K⁺-ATPase (Fig. 2D) seemed to indicate that this signaling effect of ouabain was due to the inhibition of Na⁺-K⁺-ATPase. Ouabain itself did not greatly activate ERK1/2; however, a large increase in ERK1/2 phosphorylation was promoted by inhibiting Na⁺-K⁺-ATPase by exposing the cells to a low extracellular K⁺ medium. Because both conditions (low extracellular K⁺ and high ouabain concentrations) inhibited Na⁺-K⁺-ATPase, these data suggest that the two different effects (i.e., potentiation and activation) on ERK1/2 involve, at least in part, nonoverlapping mechanisms. The activation of ERK1/2 in cardiac myocytes by ouabain was reported to be mimicked by low extracellular K⁺ (data not shown) (10), but few other studies have reported comparisons of low K⁺ and ouabain on ERK1/2 activation. Differences in the concentration dependence of ouabain in activating ERK1/2 in rat- and pig-derived cell lines correlated well with the different ouabain sensitivities of the pig and rodent Na⁺-K⁺-ATPase ₁-subunits, suggesting that the effects of ouabain were due to its interaction with Na⁺-K⁺-ATPase (11). However, because the stimulation of ERK1/2 by ouabain in various cell systems is often produced by concentrations that do not cause a large inhibition of Na⁺-K⁺-ATPase, this phenomenon has led to the hypothesis that ouabain acts as a Na⁺-K⁺-ATPase ligand rather than as an inhibitor. We are unable to make this distinction on the basis of the similar EC₅₀ and IC₅₀ values of ouabain regarding Na⁺-K⁺-ATPase inhibition and ERK1/2 potentiation, respectively. Our data cannot exclude Na⁺-K⁺-ATPase inhibition having a causal role in the modest ERK1/2 activation by ouabain, the potentiating action of ouabain when it is present with carbachol, or the activation of ERK1/2 by low extracellular K⁺.

ERK1/2 can directly and indirectly regulate Na⁺-K⁺-ATPase and/or transepithelial Na⁺ transport in a variety of epithelial cells (2, 7, 22). The -subunit can be phosphorylated by ERK in vitro on serine and threonine residues (2, 13). Some ERK-dependent effects were acute and were due to various mechanisms, including an increase in the cell surface expression of Na⁺-K⁺-ATPase -subunits (2); other effects of ERK1/2 were manifested on a longer time scale and in some cases involved alterations in the total levels of Na⁺-K⁺-ATPase subunits. The reduction (11.4 ± 1.0%, n = 3; P < 0.01) of the carbachol-initiated increases in QO₂ (an indicator of Na⁺-K⁺-ATPase activity) in U0126-treated cells does not indicate whether ERK could have had a direct or indirect (via an upstream ion channel or transporter) on Na⁺-K⁺-ATPase. However, the reduction (10.8 ± 3.8%, n = 4; P = 0.06) of nystatin-stimulated QO₂ and/or Na⁺-K⁺-ATPase activity in the U0126-treated cells suggests that ERK may have a direct effect, because nystatin exerts its stimulatory effect by removing the substrate (Na⁺) limitation to Na⁺-K⁺-ATPase that exists under basal conditions, which promotes an increase in oxidative phosphorylation to support enhanced parotid Na⁺-K⁺-ATPase activity (31, 32). Although the measurement of cell ATP levels suggested otherwise, one caveat regarding these conclusions is that these results are dependent on U0126 not affecting a component of oxidative metabolism that is unrelated to the energy-dependent activation of Na⁺-K⁺-ATPase.

We were unable to distinguish the upstream activators of ERK1/2 by 10^–5 M carbachol from those upstream signaling proteins that contributed to ERK1/2 activation by ouabain plus submaximal carbachol concentrations. Notably, it did not take long for the amplification effect to be set into motion, because the cells did not have to be pretreated with ouabain (Fig. 1B). Moreover, the potentiation effect of ouabain did not involve several important signaling events that are critical for the activation of ERK1/2 by ouabain alone in other cells (35). Thus EGFR and ROS generation did not play any role in our present findings; however, Src and PKC did contribute to our findings, as they did also when ouabain activated ERK1/2 in a variety of cells. Because the Ca²⁺ chelator BAPTA blocked the potentiating effects of ouabain on carbachol-promoted ERK1/2 activation, we checked for effects of ouabain on overall changes in carbachol-promoted [Ca²⁺]_i release and [Ca²⁺]_i homeostasis; however, ouabain itself did not cause a measurable release of Ca²⁺ or affect Ca²⁺ signaling by muscarinic receptor activation (Fig. 5). Thus ouabain did not appear to promote an increase in the muscarinic receptor activation of PLC and appeared to act downstream from the muscarinic receptor. In renal cells, ouabain produced Ca²⁺ oscillations by activating the InsP₃ receptor (InsP₃R) in an InsP₃-independent manner, and this activation appeared to involve the association of Na⁺-K⁺-ATPase with the InsP₃R (23). We were precluded from detecting these kinds of changes in our cuvette-based [Ca²⁺]_i experiments, but our data suggest that ouabain did not enhance the carbachol-initiated production of InsP₃.

In many systems in which ouabain activates ERK1/2, it appears to do so by assembling a complex of signaling proteins in caveolin microdomains (6, 16, 17, 33). Within minutes, the Na⁺-K⁺-ATPase ₁-subunit (to which ouabain binds), Src, EGFR, and ERK1/2 colocalized in caveolin-enriched vesicles. Thus one possible explanation for the present results may be that ouabain affects the colocalization and/or interactions of the relevant signaling proteins that participate in ERK1/2 activation, and its amplification effect on lower concentrations of carbachol may not involve a different roster of signaling molecules from those that participate when a larger concentration of carbachol is used in the absence of ouabain. Another possible explanation might be that ouabain does act at the level of the muscarinic receptor. Mutational studies of the M3 muscarinic receptor (the main form present in parotid acinar cells; see Ref. 34) have indicated that its coupling to PLC can be distinct from its activation of ERK/1/2 because of the different dependencies of PLC and ERK1/2 phosphorylation on receptor dimerization (26).

A variety of biological effects (e.g., differentiation, proliferation, protein expression) of the Na⁺-K⁺-ATPase inhibitor ouabain are dependent on ERK1/2 activation (6, 35). In salivary gland cells and cell lines, ERK1/2 affects a variety of responses, including the expression of ion and water channels (12, 36) and developmental events (21). In other cells, ERK also affects the expression and activity of several ion transport proteins, including Na⁺-K⁺-ATPase (2, 13, 14, 22, 37). Thus ERK1/2 and Na⁺-K⁺-ATPase each can affect each other's activation. Our findings suggest that this phenomenon can occur in a defined manner within parotid epithelial cells.

	GRANTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institute of Dental and Craniofacial Research Grant DE-10877.

	ACKNOWLEDGMENTS

 
We thank Dr. Benjamin Neel (Beth Israel Deaconess Medical Center) for the use of the Odyssey Infrared Imaging System.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. P. Soltoff, Harvard Medical School, New Research Bldg. Rm. 1030 J, 77 Ave. Louis Pasteur, Boston, MA 02115 (e-mail: ssoltoff{at}bidmc.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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