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Am J Physiol Cell Physiol 291: C1247-C1257, 2006. First published June 28, 2006; doi:10.1152/ajpcell.00593.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells

Syed J. Khundmiri,1 Melissa A. Metzler,2 Mohamed Ameen,1 Vishal Amin,1 Madhavi J. Rane,1,* and Nicholas A. Delamere2,*

Departments of 1Medicine and 2Ophthalmology, University of Louisville, Louisville, Kentucky

Submitted 28 November 2005 ; accepted in final form 30 May 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cardiotonic glycosides, like ouabain, inhibit Na+-K+-ATPase. Recent evidence suggests that low molar concentrations of ouabain alter cell growth. Studies were conducted to examine the effect of ouabain on Akt phosphorylation and rate of cell proliferation in opossum kidney (OK) proximal tubule cells. Cells exposed to 10 nM ouabain displayed increased Akt Ser473 phosphorylation, as evidenced by an increase in phospho-Akt Ser473 band density. Ouabain-stimulated Akt Ser473 phosphorylation was inhibited by pretreatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 and wortmannin), a PLC inhibitor (edelfosine), and an Akt inhibitor. Moreover, ouabain-mediated Akt Ser473 phosphorylation was suppressed by reduction of extracellular calcium (EGTA) or when intracellular calcium was buffered by BAPTA-AM. An inhibitor of calcium store release (TMB-8) and an inhibitor of calcium entry via store-operated calcium channels (SKF96365) also suppressed ouabain-mediated Akt Ser473 phosphorylation. In fura-2 AM-loaded cells, 10 nM ouabain increased capacitative calcium entry (CCE). Ouabain at 10 nM did not significantly alter baseline cytoplasmic calcium concentration in control cells. However, treatment with 10 nM ouabain caused a significantly higher ATP-mediated calcium store release. After 24 h, 10 nM ouabain increased the rate of cell proliferation. The Akt inhibitor, BAPTA-AM, SKF96365, and cyclopiazonic acid suppressed the increase in the rate of cell proliferation caused by 10 nM ouabain. Ouabain at 10 nM caused a detectable increase in 86Rb uptake but did not significantly alter Na+-K+-ATPase (ouabain-sensitive pNPPase) activity in crude membranes or cell sodium content. Taken together, the results point to a role for CCE and Akt phosphorylation, in response to low concentrations of ouabain, that increase the rate of cell proliferation without inhibiting Na+-K+-ATPase-mediated ion transport.

Na+-K+-ATPase; opossum kidney cells

THE SODIUM PUMP, Na+-K+-ATPase, is a member of the P-type ATPase family. The primary function of Na+-K+-ATPase is to maintain normal sodium and potassium gradients across the membrane of the cell. In kidney proximal tubules, Na+-K+-ATPase is localized to the basolateral membrane and establishes Na+ gradients that provide the driving force for vectorial transport of various solutes and ions, including sodium, from the tubular lumen to the renal vasculature. Regulation of proximal renal tubule sodium reabsorption is essential for whole-body sodium homeostasis, regulation of extracellular fluid volume, and blood pressure control (20, 50).

Cardiac glycosides such as ouabain selectively inhibit Na+-K+-ATPase-mediated ion transport, causing an increase in cytoplasmic sodium (3). However, cardiac glycosides may elicit other responses. Recent evidence suggests that Na+-K+-ATPase might act as a signal transducer in the sense that interaction with cardiac glycosides initiates a series of protein-protein interactions that leads to the generation of second messengers (46). In this respect, several studies have demonstrated that ouabain stimulates activation of several signaling proteins including phosphatidylinositol 3-kinase (PI3K), tyrosine kinases, the Ras-Raf-MEK pathway, and protein kinase C (26, 30, 35, 47, 51). However, there has not been a previous report of Akt phosphorylation in response to an ouabain concentration insufficient to inhibit pump activity. In some cell types, the mechanism proposed involves transactivation of epidermal growth factor receptor (EGFR) (31, 32). The physiological responses to ouabain are varied. In cardiac myocytes, ouabain causes hypertrophy (46), whereas in other cell types, ouabain alters the patterns of cell proliferation (2, 5, 6, 8, 38, 45, 51).

Plasma levels of endogenous cardiotonic glycosides (ouabain and marinobufagenin), normal products of mammalian adrenal glands, are increased in various disease states such as essential hypertension (33, 34), chronic renal failure (23), and experimental uremia (16). It has been reported that ouabain (10 nM) activates extracellular signal-regulated kinase (ERK) and stimulates cell proliferation in cultured primary proximal tubular cells (8). Since Akt (protein kinase B) is known to influence cell proliferation, we hypothesized that ouabain might induce cell proliferation through an Akt-mediated pathway. Akt is present in the cytosol of unstimulated cells in a low-activity conformation. Upon cellular stimulation, Akt translocates to the membrane and undergoes phosphorylation at Thr308 and Ser473 to assume its active conformation. Akt activation occurs when growth factors or other ligands stimulate PI3K, thereby causing release of 3'-phosphorylated phosphoinositides that regulate Akt in at least two ways (41). First, phosphoinositides bind directly to the pleckstrin homology domain causing translocation of Akt to the plasma membrane (12). Second, phosphoinositides activate the phosphoinositide-dependent kinases PDK1 and PDK2 that phosphorylate Thr308 and Ser473, respectively (2, 41). The active form of Akt exerts its anti-apoptotic effect by phosphorylating a number of substrates, including BAD (7), caspase 9 (4), Ask1 (25), and the forkhead transcription factors FOXO (27) and FOXO3a (22). Upon phosphorylation, a number of Akt substrates bind adaptor protein 14-3-3 and regulate apoptosis. Apart from regulating cell survival, Akt has been shown to regulate a number of cellular functions including motility, growth, and metabolism (41). Studies to examine the effects of ouabain on Akt were conducted using OK cells, an opossum kidney proximal tubule cell line. Ouabain was found to phosphorylate Akt by a mechanism dependent on PI3K, PLC, and calcium. Additionally, inhibition of Akt suppressed ouabain-induced cell proliferation. Importantly, the effects were observed at an ouabain concentration too low to inhibit Na+-K+-ATPase-mediated ion transport.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Ouabain, 8-(diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8), SKF96365, and cyclopiazonic acid (CPA) were purchased from Sigma (St. Louis, MO). Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, wortmannin, LY294002, BAPTA-AM, and edelfosine (ET18-OCH3, an inhibitor of phosphatidylinositol-dependent PLC) were purchased from Calbiochem-EMD Biosciences (San Diego, CA). Fura-2 AM and Pluronic F127 were purchased from Invitrogen (Carlsbad, CA). Antibodies against phospho-Ser473-Akt and total Akt were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against Na+-K+-ATPase {alpha}2 (AB9094) and {alpha}3 (A273) subunits were purchased from Chemicon International (Temecula, CA) and Sigma-RBI (Natick, MA), respectively. HRP-linked secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were purchased from Sigma, unless otherwise specified.

Cell culture. The opossum kidney (OK) cells, a continuous cell line derived from Virginia opossum renal proximal tubule (28) was a kind gift from Dr. Steven Scheinman (State University of New York Health Sciences Center, Syracuse, NY). OK cells were maintained in minimal essential medium with Earle's salts (EMEM) supplemented with 10% FCS, and 1% penicillin/streptomycin. Cell culture and all other studies were carried out at 37°C in a humidified atmosphere of 95% air-5% CO2. The cells were fed twice a week and split once a week at a 1:4 ratio. All experiments were carried out using cells at 90–95% confluence. Cells grown on 6-well culture plates were washed with serum-free medium 24 h before use. For measurement of calcium, cells were cultured on glass slides.

Western blot analysis. The cells were placed in ice-cold lysis buffer containing 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 20 mM NaF, 1 mM EGTA, 1 mM EDTA, 5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µl/ml phosphatase inhibitor cocktail 1, 0.5% NP-40, and 1% Triton X-100. The cell lysate was homogenized by passing them through 271/2 gauge needles, and then centrifuged at 20,000 g at 4°C. The supernatant proteins were separated by 10% SDS-PAGE, and then transferred to nitrocellulose membranes. The nitrocellulose membranes were incubated in 20 mM Tris·HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20 (TTBS) containing 5% milk at room temperature for 1 h to inhibit nonspecific binding, followed by overnight incubation at 4°C with anti-phospho-Ser473-Akt antibodies at 1:1,000 dilution in TTBS containing 5% milk. After being washed, the nitrocellulose membranes were exposed to peroxidase-labeled secondary antibodies at 1:2,000 dilution in TTBS containing 5% milk. Bands were detected by chemiluminescence (New England Biolabs), visualized on X-ray film, and quantified by densitometry by using a Personal Densitometer SI (Molecular Dynamics).

Membrane isolation. The cells were washed twice with PBS and homogenized in ice-cold 50 mM mannitol, 5 mM Tris·HCl, pH 7.4. The lysate was homogenized using a high-speed homogenizer (3 strokes, 15 s), incubated with 10 mM MgCl2 for 20 min on ice, followed by centrifugation at low speed (3,000 rpm for 10 min) to remove cell debris. The supernatant was centrifuged at 20,000 g for 20 min. The pellet, containing crude membrane material, was resuspended in the homogenizing buffer. Crude membrane preparations were analyzed for the expression of Na+-K+-ATPase {alpha}2 and {alpha}3 by Western blot analysis.

Determination of K+-dependent pNPPase activity. Ouabain-sensitive pNPPase activity was measured as an index of Na+-K+-ATPase activity. pNPPase activity was measured in crude membranes as described previously (28) following the method of Hird et al. (17), with slight modifications, as described by Tran and Farley (43). Briefly, cleavage of p-nitrophenyl phosphate to p-nitrophenol was quantified in the presence of a high concentration of potassium. Twenty microliters of the crude membrane preparation (25–30 µg protein) was added to 0.9 ml assay buffer (30 mM histidine, 150 mM KCl, 20 mM MgCl2, 2 mM EGTA, 10 mM p-nitrophenyl phosphate, pH 7.4, containing 10 nM or 10 µM ouabain). The assay mixture was incubated for 15 min at 37°C. The reaction was stopped by the addition of 100 µl of 5N NaOH containing 0.1% Triton X-100. A standard curve was constructed by serial dilutions of p-nitrophenol. The samples and the standards were centrifuged at 1,500 g for 15 min before spectrophotometric analysis. Absorbance of the supernatant was measured at 410 nm. Ouabain-sensitive pNPPase activity was defined as the difference between activities measured in the presence or absence of 10 mM ouabain, an ouabain concentration sufficient to cause maximal inhibition. The assay was run in triplicate and activity expressed as micromoles of p-nitrophenol released per milligram protein per hour.

Ouabain-sensitive 86Rb uptake. Ouabain-sensitive 86Rb uptake was measured as described previously (24) as an index of Na+-K+-ATPase-mediated ion transport. OK cells were pretreated with 5 µM monensin for 30 min. The cells were exposed to 10 nM or 10 µM ouabain for 5 min before adding a trace amount of 86Rb (~1 µCi/ml 86RbCl) in DMEM without serum. Uptake was carried out for 10 min, such that total ouabain treatment time was 15 min, after which the cells were washed 5–6 times with ice cold PBS. Half the cells received ouabain (final concentration 1 mM) added 15 min before the start of 86Rb uptake. The cells were lysed overnight in 0.5N NaOH containing 0.1% Triton-X 100 at 37°C. An aliquot (100 µl) of the lysate was used to measure radioactivity. The difference between 86Rb uptake measured in the presence of 1 mM and 10 nM or 10 µM of ouabain was used as a measure of Na+-K+-ATPase-mediated transport activity. Uptake data are expressed as nanomoles rubidium (86Rb) accumulated per milligram of protein per minute.

Measurement of cell sodium. Cell sodium was measured according to the method described by Hou and Delamere (18). The cell monolayers were washed with ice-cold isotonic magnesium chloride solution (100 mM MgCl2, adjusted to pH 7.4 with Tris base). The magnesium chloride solution was removed and 200 µl of 30% nitric acid was added to each well to digest the cells. After this, 1.8 ml of deionized water was added to each well, and the sodium content of the diluted cell lysates was measured by using an atomic absorption spectrophotometer (Perkin-Elmer, Norwalk, CT) at a wavelength of 566.5 nm.

Measurement of cytoplasmic calcium. Cytoplasmic calcium concentration was measured as described by Okafor et al. (37) by using fura-2 AM, the calcium-sensitive fluorescent dye. Briefly, cultured OK cells attached to glass coverslips were loaded with fura-2 AM for 30 min at 37°C. Fura-2 AM was dissolved in 20% Pluronic F127, then added to Krebs solution (120 mM NaCl, 4.8 mM KCl, 5.5 mM dextrose, 3.12 mM NaH2PO4, and 12.48 mM Na2HPO4. The pH was adjusted to 7.4, following which, 1.2 mM MgSO4 and 0.54 mM CaCl2 were added slowly to avoid precipitate formation) at a final concentration of 5 µM fura-2 AM and 0.04% Pluronic F127. After loading, the cells were washed with Krebs solution. The glass coverslip was placed on the stage of a microscope (Zeiss, Thornwood, NY) equipped with a digital fluorescence imaging system (Attofluor; Atto Instruments, Rockville, MD). The cells were continuously superfused with Krebs solution and maintained at 37°C using a heated microscope stage. The emission wavelength was 520 nm. Alternating excitation wavelengths of 334 and 380 nm were used, and fluorescence was continuously quantified. To calibrate the fluorescence signal, the cells were permeabilized at the end of each experiment by adding 10 µM ionomycin to the calcium-containing superfusate to establish the maximum signal. Then, 5 mM EGTA was added to the superfusate to obtain the minimum signal. In each experiment, data were averaged from 30–40 cells. The calcium concentration was computed according to the formula supplied by the fura-2 AM manufacturer (Molecular Probes). Peak and plateau values of agonist calcium responses were calculated as the difference between either the peak or plateau value and the baseline.

Measurement of cell proliferation. The rate of cell proliferation was determined using a CellTiter 96 AQueous One assay (Promega). Briefly, cells were cultured in a 96-well plate (100 µl of culture medium/well). After the cells adhered to the plates (6 h), they were exposed to 10 nM or 10 µM ouabain as specified. The rate of cell proliferation, as reflected by metabolism of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, was determined by adding CellTiter 96 AQueous One solution reagent (20 µl) to each culture well. After incubation for 3 h, the absorbance at 490 nm was measured to quantify the amount of formazan product (8). Cells in the outer two lanes of the plate (16 wells) were washed twice with PBS and lysed in 0.5% Triton X-100 to determine the amount of protein. The data are expressed as optical density (OD at 490 nm) per milligram of protein.

Protein determination. Protein concentration was determined using a bicinchoninic acid protein kit (Sigma) using BSA as the standard.

Statistics. Data are shown as means ± SE. The n values shown represent the number of independent experiments. Each experiment was done in triplicate. P values were calculated using SigmaStat software utilizing Student's t-test or by one-way ANOVA, followed by the Bonferroni analysis using GraphPad Prism software. A P value <0.05 was a priori considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of ouabain on Akt Ser473 phosphorylation. OK cells were treated for 15 min with ouabain, ranging from 1 nM to 10 µM. Akt phosphorylation in the ouabain-treated cells was detected by Western blot analysis using anti-phospho-Akt Ser473 and anti-Akt antibodies. As shown in Fig. 1A, ouabain treatment significantly increased the band density of phospho-Akt. The phosphorylation of Akt was maximal at 10 nM ouabain. Treatment with 1 µM ouabain decreased Akt phosphorylation compared with 10 and 100 nM ouabain treated cells. Total Akt immunoblot band density was not altered by ouabain treatment. To determine the time course of Akt phosphorylation, cells were treated with 10 nM ouabain for 0–360 min, and cell lysates were immunoblotted with anti-phospho-Akt Ser473 and anti-Akt antibodies. As shown in Fig. 1B, maximal Akt phosphorylation was observed after 15 min ouabain treatment.


Figure 1
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  		Fig. 1. Effect of ouabain on Akt phosphorylation. OK cells were treated for 15 min with different concentrations of ouabain (A) or with 10 nM ouabain for different times ranging from 5–360 min (B). Cell lysates were obtained, then subjected to 10% SDS-PAGE, transferred to membranes, and probed for phospho-Akt-Ser473 (top). The blots were stripped and reprobed for total Akt (bottom). A representative Western blot is shown. Phospho-Akt and total Akt band densities were analyzed by densitometry and the data (means ± SE from three independent experiments) are presented as ratio of phospho-Akt Ser473 to total Akt band density. *P < 0.05 by Student's t-test.

 
Effect of ouabain on pNPPase activity. Ouabain-sensitive pNPPase activity, an index of Na+-K+-ATPase activity, was measured in a crude membrane preparation isolated from OK cells that had been treated for 15 min with either 10 nM or 10 µM ouabain. As shown in Fig. 2A, 10 nM ouabain did not significantly alter pNPPase activity; however, ouabain at 10 µM inhibited pNPPase activity by about 60%. Ouabain-sensitive 86Rb uptake was measured as an index of Na+-K+-ATPase-mediated ion transport in intact cells. As shown in Fig. 2B, 10 µM ouabain significantly decreased ouabain-sensitive 86Rb uptake. In contrast, 10 nM ouabain significantly stimulated ouabain-sensitive 86Rb uptake (Fig. 2B). In a separate group of cells, sodium content was measured by atomic absorption spectroscopy. Cell sodium was not significantly altered following treatment with 10 nM ouabain for either 15 min or 4 h (Fig. 2C). In contrast, 10 µM ouabain elicited a significant increase in cell sodium detectable at both the time points.


Figure 2
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  		Fig. 2. The effect of ouabain on Na+-K+-ATPase activity and sodium content. A: Na+-K+-ATPase activity was measured in crude membranes obtained from OK cells that had been treated for 15 min with either 10 nM or 10 µM ouabain. Na+-K+-ATPase activity, measured as ouabain-sensitive pNPPase activity, is presented as means ± SE of results from three independent experiments. B: intact cells were incubated with or without 10 nM or 10 µM ouabain for 5 min at 25°C, and subsequently, ouabain-sensitive 86Rb uptake was measured over a 10-min period, such that total time of ouabain treatment was 15 min. Each bar represents means ± SE from six independent experiments performed in triplicate. C: in separate experiments, cell sodium content was measured in OK cells that had been treated with either 10 nM or 10 µM ouabain for 15 min or 4 h. Sodium content is presented as the means ± SE of results from three independent experiments. *P < 0.05 compared with control using Student's t-test.

 
Role of PI3K in ouabain-stimulated Akt Ser473 phosphorylation. Since Akt is a downstream target of PI3K (41), OK cells were pretreated with PI3K inhibitors LY294002 (5 µM) or wortmannin (100 nM), before stimulation with ouabain (10 nM). Both PI3K inhibitors suppressed the ouabain-induced increase in phospho-Akt band density. Pretreatment of OK cells with the Akt inhibitor (50 µM) prevented ouabain-stimulated Akt Ser473 phosphorylation (Fig. 3). This Akt inhibitor has been shown to interact in a complementary manner with the positively charged pocket formed by beta1-beta2 and beta3-beta4 loops of the pleckstrin homology domain of Akt, thereby preventing the binding of phosphatidylinositol trisphosphate and translocation of Akt to membrane (19).


Figure 3
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  		Fig. 3. Role of phosphatidylinositol 3-kinase (PI3K). OK cells were treated for 15 min with 10 nM ouabain in presence of pharmacological inhibitors of Akt (Akt inhibitor; 50 µM) and PI3K (LY294002; 5 µM and wortmannin; 100 nM). Cell lysates were obtained, then subjected to 10% SDS-PAGE, transferred to membranes, and probed for phospho-Akt-Ser473 (top). The blots were stripped and reprobed for total Akt (bottom). A representative Western blot is shown. Phospho-Akt and total Akt band densities were analyzed by densitometry and the data (means ± SE from three independent experiments) are presented as ratio of phospho-Akt Ser473 to total Akt band density. *P < 0.05 by Student's t-test.

 
Role of calcium and PLC in ouabain-stimulated Akt Ser473 phosphorylation. To examine the role of calcium in ouabain-mediated phosphorylation of Akt, EGTA (1 mM) was added to chelate calcium in the bathing medium, or BAPTA-AM (20 µM) was added to buffer intracellular calcium. In addition, OK cells were exposed to 10 nM ouabain in the presence of TMB-8 (100 µM), an inhibitor of endoplasmic reticulum (ER) calcium store release (10). In each case, the ouabain-mediated increase in phospho-Akt band density was suppressed (Fig. 4, A and B). These findings suggest that phosphorylation of Akt by ouabain may require entry of extracellular calcium, as well as calcium released from ER stores. To examine the requirement for calcium entry via store-operated calcium channels (SOCCs), cells were exposed for 15 min to 10 nM ouabain in the presence or absence of the SOCC inhibitor SKF96365 (25 µM). Pretreatment with SKF96365 prevented the ouabain-induced increase in phospho-Akt band density (Fig. 4C). SKF96365 also prevented Akt phosphorylation at all ouabain concentrations (data not shown). The ability of TMB-8 to suppress the phospho-Akt response to ouabain suggested the possible involvement of calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores. Since IP3 is generated by PLC, studies were performed to determine whether PLC plays a role in Akt Ser473 phosphorylation. OK cells were treated for 15 min with 10 nM ouabain in the continued presence or absence of 30 µM edelfosine (ET18-OCH3), an inhibitor of phosphatidylinositol-dependent PLC. As shown in Fig. 5, edelfosine inhibited the ouabain-induced increase in phospho-Akt band density.


Figure 4
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  		Fig. 4. Role of calcium. A: to examine the role of calcium, OK cells were treated for 15 min with 10 nM ouabain in the presence of TMB-8 (100 µM), an inhibitor of ER calcium store release. B: OK cells were treated for 15 min in the presence or absence of BAPTA-AM (20 µM) or EGTA (1 mM). In the Western blots, V denotes vehicle, B denotes BAPTA-AM, and E denotes EGTA pretreatment. C: OK cells were treated for 15 min with 10 nM ouabain in presence of SKF96365 (25 µM), an inhibitor of store-operated calcium channels. Cell lysates were obtained, then subjected to 10% SDS-PAGE, transferred to membranes, and probed for phospho-Akt-Ser473 (top). The blots were stripped and reprobed for total Akt (bottom). A representative Western blot is shown. Phospho-Akt and total Akt band densities were analyzed by densitometry and the data (means ± SE from three independent experiments) are presented as ratio of phospho-Akt Ser473 to total Akt band density. *P < 0.05 by Student's t-test.

 

Figure 5
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  		Fig. 5. Role of PLC. OK cells were treated for 15 min with 10 nM ouabain in presence of the phosphoinositol-dependent PLC inhibitor edelfosine (ET18-OCH3; 30 µM). Cell lysates were obtained, then subjected to 10% SDS-PAGE, transferred to membranes, and probed for phospho-Akt-Ser473 (upper panel). The blots were stripped and reprobed for total Akt (bottom). A representative Western blot is shown. Phospho-Akt and total Akt band densities were analyzed by densitometry and the data (means ± SE from three independent experiments) is presented as ratio of phospho-Akt Ser473 to total Akt band density. *P < 0.05 by Student's t-test.

 
Effect of ouabain on cytoplasmic calcium concentration. Cytoplasmic calcium concentration was measured in cultured OK cells preloaded with fura-2 AM and then superfused with Krebs solution. When 10 nM ouabain was added to the superfusate, cytoplasmic calcium did not change significantly from the basal values (Fig. 6A). Since the SOCC antagonist SKF96365 suppressed the Akt phosphorylation in response to 10 nM ouabain, studies were conducted to test whether ouabain alters the pattern of capacitative calcium entry (CCE). To measure CCE, cells were superfused with calcium-free buffer, then exposed to 15 µM CPA. Under these conditions, depletion of ER calcium stores leads to opening of SOCCs, and the introduction of 2.5 mM calcium to the superfusate causes a sharp increase in cytoplasmic calcium concentration ([Ca2+]i). In cells exposed to 10 nM ouabain, added 15 min before the start of the experiment, the magnitude of the increase in [Ca2+]i during CCE was significantly higher than that observed in vehicle-treated cells (Fig. 6B). Of note, the baseline [Ca2+]i was higher in ouabain-treated cells, although it was not significantly different than the control cells. Furthermore, CPA released Ca2+ was significantly higher in ouabain-treated cells. The rise in cytoplasmic calcium concentration caused by restoration of external calcium was prevented by pretreatment of cells with the SOCC inhibitor SKF96365 (Fig. 6B). As an estimate of calcium store size, [Ca2+]i was measured in ouabain-treated cells as ATP-releasable calcium. Exposure to ATP caused a significantly higher transient increase in [Ca2+]i in ouabain-treated cells compared with control cells (Fig. 6C).


Figure 6
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  		Fig. 6. The influence of ouabain on cytoplasmic calcium concentration. A: OK cells were loaded with fura-2 AM and cytoplasmic calcium concentration was recorded for 5 min in Krebs solution to obtain a baseline (left). Then, 10 nM ouabain was added to the superfusate. The bar graph (right) shows cytoplasmic calcium concentration measured 1 min before and 5 min after the addition of 10 nM ouabain (means ± SE nM, n = 10 independent experiments; in each experiment, data from 30–40 individual cells was averaged and considered as n = 1). B: to measure capacitative calcium entry, OK cells were loaded with fura-2 AM, and superfused with calcium-free Krebs solution (left). After 5 min, 10 µM cyclopiazonic acid (CPA) was added to the superfusate, and calcium concentration was recorded until a new baseline was established. Then, 2.5 mM calcium was added to the superfusate. One group of cells was treated with 10 nM ouabain during the de-esterification phase (15 min before the start of calcium measurement). Arrows show the time points at which CPA or Ca2+ was added (solid arrows for control, SKF96365, and SKF96365 + ouabain samples, and broken arrows for ouabain alone samples). The bar graph (right) shows cytoplasmic calcium concentration (means ± SE nM, n = 5 independent experiments; in each experiment data from 30–40 individual cells was averaged and considered as n = 1) measured at peak after the addition of calcium to the superfusate; *P < 0.05, significant difference compared with the control peak height. C: OK cells were loaded with fura-2 AM, and superfused with Krebs solution for 2 min to obtain a baseline cytoplasmic calcium concentration (left). After 2 min, 50 µM ATP was added to the superfusate, causing a transient increase in cytoplasmic calcium. One group of cells was treated with 10 nM ouabain during the de-esterification phase (15 min before the start of calcium measurement). The bar graph shows peak cytoplasmic calcium concentration after addition of ATP (means ± SE nM, n = 5 independent experiments; in each experiment, data from 30–40 individual cells was averaged and considered as n = 1). *P < 0.05, significant difference by Student's t-test.

 
Effect of ouabain on cell proliferation. To determine the effect of ouabain on the rate of cell proliferation, OK cells grown on a 96-well plate were treated with either 10 nM or 10 µM ouabain, following attachment of cells to the plate (about 6 h). After specified time periods, the rate of cell proliferation was examined. In the presence of 10 nM ouabain, the rate of cell proliferation observed at 12 h and 24 h was significantly increased. After 48 h treatment, the rate of proliferation diminished as the cells reached confluence (Fig. 7A). In contrast, 10 µM ouabain decreased the rate of cell proliferation in a time-dependent manner. To examine the possible link between Akt phosphorylation and calcium on the rate of cell proliferation in response to 10 nM ouabain, cells were treated with 10 nM ouabain for 24 h in the presence or the absence of the Akt inhibitor (50 µM), BAPTA-AM (20 µM), CPA (15 µM), or SKF96365 (25 µM). Pretreatment with the Akt inhibitor (Fig. 7B), BAPTA-AM, CPA, or SKF96365 (Fig. 7C) abolished the ouabain-stimulated increase in the rate of cell proliferation. Added alone, the Akt inhibitor and BAPTA-AM did not significantly alter the rate of cell proliferation. However, CPA and SKF96365 alone significantly decreased the rate of cell proliferation.


Figure 7
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  		Fig. 7. The effect of ouabain on cell proliferation. A: OK cells were treated with 10 nM or 10 µM ouabain after the cells were attached to the 96-well plate for 12, 24, or 48 h. Cell proliferation was measured as described in EXPERIMENTAL PROCEDURES. Each data point represents means ± SE from 24 independent experiments. B: OK cells were treated for 24 h with 10 nM ouabain in the presence or absence of Akt inhibitor. C: OK cells were treated for 24 h with 10 nM ouabain in the presence or absence of BAPTA-AM (20 µM), CPA (15 µM), or SKF96365 (25 µM). Cell proliferation was measured as described in EXPERIMENTAL PROCEDURES. Each bar represents means ± SE of results from 12 independent experiments. *P < 0.05, compared with control using one-way ANOVA, followed by the Bonferroni analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we report for the first time that exposure of OK cells to a low concentration of ouabain (10 nM) resulted in increased Akt phosphorylation, as evidenced by an increase in phospho-Akt band density, and increased the rate of cell proliferation. Importantly, inhibition of Akt phosphorylation abolished the effect of 10 nM ouabain on cell proliferation. Indirect evidence, reported by Zhou et al. (51), suggests that Akt may be activated by ouabain in LLC-PK1 cells, a porcine proximal tubule cell line. These authors employed a relatively high concentration of ouabain, sufficient to inhibit Na+-K+-ATPase-mediated ion transport, and observed activation of PI3K, as well as Akt activation. On the basis of the ability of LY294002, wortmannin, and edelfosine to suppress Akt phosphorylation by ouabain, the results from the present study suggest that phosphorylation of Akt in ouabain-treated OK cells is dependent upon PI3K and PLC.

Although ouabain is recognized primarily as a specific inhibitor, inhibition of Na+-K+-ATPase-mediated ion transport is not required for ouabain to stimulate Akt phosphorylation. An increase in phospho-Akt was observed at a ouabain concentration of 10 nM, a concentration that was not sufficient to inhibit Na+-K+-ATPase activity, as judged by measurements of ouabain-sensitive pNPPase activity. An earlier report by Pedemonte et al. (39), similarly demonstrated that 10 nM ouabain had no effect on Na+-K+-ATPase activity. 86Rb uptake studies were conducted to examine Na+-K+-ATPase-mediated ion transport function in intact OK cells. At 10 nM ouabain did not inhibit, but instead caused a significant increase in 86Rb uptake, indicating stimulation of the Na-K pump. The response is consistent with reports of Na-K pump stimulation by a low concentration of ouabain in cardiac myocytes (13) and in rat renal epithelial (NRK 52E) cells (11). The mechanism by which a low concentration of ouabain stimulates 86Rb uptake remains to be elucidated.

Studies by Askari and coworkers have presented evidence that suggests Na+-K+-ATPase in cardiac myocytes acts as a signal transducer, in the sense that interaction of Na+-K+-ATPase with extracellular ouabain leads to activation of the ERK/MAP kinase pathway (26, 31, 32, 35, 44–46, 48). Here we provide evidence for Akt phosphorylation in cells exposed to 10 nM ouabain. Akt is a known downstream target of PI3K (41). This fits with the observation that inhibition of PI3K by LY294002 and wortmannin suppresses ouabain-mediated phosphorylation of Akt.

Our data points to a role for calcium in the Akt phosphorylation response to ouabain. Ouabain is known to alter cytoplasmic calcium under certain conditions. For example, Aizman and coworkers (1) demonstrated that ouabain elicits calcium oscillations in proximal tubule cells. However, the calcium response pattern is somewhat variable since in LLC-PK1 cells, 100 nM ouabain was found to elicit a detectable calcium rise in ~40% of the cells, and <1% of the cells displayed calcium oscillations (49). Findings in the present study suggest that the phosphorylation of Akt is dependent upon extracellular calcium, since ouabain-mediated phosphorylation of Akt was suppressed under conditions where the concentration of extracellular calcium was reduced. BAPTA-AM, a chelator of cytoplasmic calcium, also suppressed the Akt response to ouabain. In vascular smooth muscle cells, Akt activation induced by angiotensin II is similarly suppressed by extracellular calcium depletion, and it has been suggested that the mechanism involves the generation of arachidonic acid metabolites, following the activation of calcium-dependent phospholipase A2 (29). Phospho-Akt responses to ouabain in OK cells were also suppressed by edelfosine, a PLC inhibitor, and by TMB-8, a nonspecific intracellular calcium inhibitor that can prevent release of calcium from IP3-sensitive cytoplasmic stores (10). Edelfosine is known to inhibit growth factor-induced activation of Akt in the A431 and HeLa carcinoma cell lines (42). The ability of edelfosine and TMB-8 to inhibit the ouabain response suggests that the response mechanism may involve the generation of IP3 and the subsequent release of cytoplasmic calcium stores. However, 10 nM ouabain-treated cells loaded with fura-2 AM did not elicit a detectable cytoplasmic calcium increase. In contrast, 10 nM ouabain significantly increased the magnitude of CCE, which was suppressed by pretreatment with SKF96365 and increased the magnitude of calcium rise elicited by ATP. It is noteworthy that ouabain did not significantly alter the baseline cytoplasmic calcium in ouabain-treated cells. However, we cannot rule out an increase in baseline calcium that is small compared with the magnitude of variability in the calcium measurement. Nor can we rule out a localized calcium increase. The mechanism underlying the effect of 10 nM ouabain on CCE remains a subject for further study. On the basis of the current experiments it seems likely that SOCC-mediated calcium entry is increased, perhaps as a result of altered SOCC function in ouabain-treated cells. CCE also could increase as the consequence of impaired calcium export. In other cells, ouabain treatment has been proposed to diminish the ability to conduct Na+/Ca+ exchanger-mediated calcium export (14). This is not likely to explain the ouabain response in OK cells, a cell type that has very low level of Na+/Ca2+ exchanger expression (21). Moreover, the 10 nM concentration of ouabain that stimulated CCE-mediated calcium entry did not detectably alter cell sodium, suggesting that no change occurs in the gradient that provides the driving force for Na+/Ca2+ exchange.

The apparent stimulation of SOCC-mediated calcium entry by 10 nM ouabain fits the observed sensitivity of the Akt response to SKF96365, an inhibitor of SOCCs. In the presence of 25 µM SKF96365, ouabain did not detectably increase the phosphorylation of Akt, suggesting that calcium entry occurs, in part, via SOCCs. The same concentration of SKF96365 also inhibited CCE. It is noteworthy that ouabain is reported to elicit activation of store-operated calcium channels in astrocytes (15). While there is evidence pointing toward disturbance of calcium dynamics, following ouabain exposure in cells that express more than one {alpha} isoform of Na+-K+-ATPase (14, 15), it has been shown that similar alterations in calcium dynamics, and enhanced heart muscle contractility in response to ouabain, occur in transgenic mice expressing ouabain-sensitive {alpha}1 and ouabain-resistant {alpha}2 isoforms of Na+-K+-ATPase (9). OK cells express only the {alpha}1 isoform of the Na+-K+-ATPase catalytic subunit. Therefore, the most likely binding site for ouabain in OK cells is the Na+-K+-ATPase {alpha}1 subunit, suggesting that interaction between {alpha}1 and ouabain initiates a chain of events that leads to Akt activation. Consistent with an earlier report by Pierre et al. (40), Western blot analysis failed to detect Na+-K+-ATPase {alpha}2 or {alpha}3 subunits (data not shown). However, we are not able to rule out the possibility that these isoforms are present in small amounts.

Plasma levels of ouabain-like compounds (OLC) have been shown to increase in several disease states. Harwood and colleagues (16) have shown that plasma OLC levels are elevated in mild experimental uremia. Manunta and coworkers (33, 34) demonstrated that a ouabain-like factor is increased in the plasma of individuals with essential hypertension, and OLC levels are increased in rats with reduced renal mass-saline hypertension (47). The physiological significance of increased OLC remains to be determined, but the results of the present study indicate the potential for cardiac glycosides to alter the pattern of cell growth.

The influence of ouabain on cell growth has been difficult to define. Several investigators have proposed that ouabain causes cell proliferation and hypertrophy (8, 35) but, in contrast, others have shown that ouabain treatment leads to apoptosis (38, 44). This may reflect different patterns of response according to the concentration of ouabain. Here, we report that 10 nM ouabain significantly stimulates Akt Ser473 phosphorylation and the rate of cell proliferation, without inhibiting Na+-K+-ATPase activity, and without altering cellular sodium content. Taken together, the evidence suggests that the effects of 10 nM ouabain on Akt and cell proliferation are independent of Na+-K+-ATPase-mediated ion transport inhibition, but are linked to altered cell calcium entry and possibly calcium store size. In keeping with this notion, BAPTA-AM, CPA, and SKF96365 all were found to suppress the ability of ouabain to increase the rate of cell proliferation. It is noteworthy that 10 nM ouabain stimulated Na+-K+-ATPase-mediated ion transport. It remains to be determined whether such stimulation was linked to the Akt response or altered proliferation rate.

In summary, the findings demonstrate phosphorylation of Akt by ouabain in OK cells. The mechanism of Akt phosphorylation is calcium dependent and may involve an increase in CCE. The findings suggest a link between Akt phosphorylation and the effect of ouabain on cell proliferation. The findings support the notion that ouabain is able to change the pattern of cell growth at concentrations significantly lower than the concentration required to inhibit Na+-K+-ATPase-mediated ion transport. Further studies are required to delineate the signaling pathways activated by higher concentrations of ouabain from the pathways activated at lower ouabain concentrations.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
The work was supported by a Fellowship Grant from The American Heart Association, Ohio Valley Affiliate (to S. J. Khundmiri), Scientist Development Grant (to S. J. Khundmiri and M. J. Rane), National Eye Institute Grant EY-040414 (to N. A. Delamere), and a Project Grant from the J. Graham Brown Cancer Center, University of Louisville (to N. A. Delamere).


   ACKNOWLEDGMENTS
 
We thank Dr. Eleanor Lederer, Professor of Medicine, University of Louisville, for critical reading of the manuscript. We also thank Nina Lesousky for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Khundmiri, Kidney Disease Program, Univ. of Louisville, 570 S. Preston St. South POD 102, Louisville, KY 40202 (e-mail: syed.khundmiri{at}louisville.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.

* M. J. Rane and N. A. Delamere contributed equally to this work. Back


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