Aldosterone induces Ras methylation in A6 epithelia

N. F. Al-Baldawi, J. D. Stockand, O. K. Al-Khalili, G. Yue, and D. C. Eaton

Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Aldosterone increases Na+ reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na+ channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na+ transport; 2) one target for methylation is p21ras; and 3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity.

protein methylation; sodium transport; A6 cells; epithelial transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALDOSTERONE IS THE MOST IMPORTANT systemic modulator of apical Na+ permeability in mammalian collecting tubule principal cells. The final effect of this hormone is to increase the amount of Na+ uptake by Na+ channels in the apical membranes of transporting cells; however, the mechanisms by which aldosterone increases Na+ transport are not at all clear, and many of the proposed mechanisms are controversial. The aldosterone-mediated increase in Na+ transport occurs in two phases: an initial phase that increases transport approximately fivefold in the first 4 to 6 h, and a late phase that requires 12-48 h and increases transport another three- to fourfold. How the early and late phases are related is unclear. In this report, we will concentrate on the initial phase.

Little is known about the cellular mechanisms of aldosterone's action except that gene expression and protein synthesis are required and that the final result is an increase in Na+ transport (5, 13, 32). Originally, because of the observation that protein synthesis was required for aldosterone to increase Na+ transport, it was postulated that aldosterone induced Na+ channel synthesis and insertion. However, earlier studies in A6 cells suggest that aldosterone increases Na+ entry at the apical membrane by changing the activity of channels that are already present in the apical membrane and not by increasing the number of channels (19). Data obtained with biochemical methods support this observation, indicating that the number of channels in the apical membrane does not increase in the presence of aldosterone (at least in the first 2-4 h when the increase in Na+ transport is most dramatic) (13, 22). However, in rat cortical collecting tubules and A6 cells, aldosterone appears to increase the number of Na+ channels in the apical membrane based on single channel and noise analysis (16, 33). Similar conclusions were reached from studies in toad urinary bladder and A6 cells with the use of fluctuation analysis of amiloride-induced current noise (3, 34). Additionally, Xenopus laevis oocytes that were injected with RNA from tissue that had been treated with aldosterone for 3-24 h expressed more channel activity, suggesting that aldosterone increases the transcription of the Na+ channel gene itself (30). Thus it appears that depending on the experimental approach or system used to obtain the data, the answer to the question of whether aldosterone increases Na+ transport by increasing the transcription and translation of the Na+ channel itself remains unresolved.

However, it is now possible to directly address this question because of the identification of the genes that encode epithelial Na+ channel (ENaC) (9). Recently, Middleton et al. (31) directly addressed in A6 cells the question of whether aldosterone increases mRNA levels and/or protein levels of any or all of the three ENaC subunits. Their data show that increases in short-circuit current after 4 h exposure to aldosterone can occur without corresponding increases in mRNA or protein levels for alpha -, beta -, or gamma -ENAC. These results are consistent with the findings by Marunaka et al. (30), who measured mRNA levels of ENaCs in kidney cells of rats whose aldosterone levels had been changed either by diet or exposure to corticosteroids. No differences for beta - or gamma -ENaC message were found between rats on a normal vs. a low-salt diet, and only small increases in alpha -xENaC message were observed. Thus in renal tissues (including A6 cells), the electrophysiological evidence varies, but the biochemical evidence all supports the idea that aldosterone (in the first 2-4 h) modulates the activity of preexisting channels rather than promoting the synthesis or insertion of new channels.

There have been many suggestions about potential aldosterone-induced posttranslational modifications, but in the context of our previous results (4, 46, 47), one is particularly interesting. Sariban-Sohraby et al. (38) demonstrated that the amount of Na+ transport that could be measured in apical membrane vesicles obtained from A6 cells, a Na+-transporting, distal-nephron cell line, was markedly enhanced by prior application of agents that methylate membrane proteins. Because they also demonstrated that application of aldosterone leads to the methylation of membrane protein and lipid, their suggestion was that intracellular methyltransferases induced by aldosterone could be responsible for the methylation and, therefore, modulate the Na+ channel protein. As a posttranslational modification, methylation is analogous to phosphorylation (for reviews, see Refs. 2, 10, 36, and 48). Highly specific methyltransferases promote the methylation of proteins at specific consensus sites, and much less specific esterases promote demethylation of the proteins. The difference between phosphorylation and methylation is that methylation is in general more stable and, therefore, can be used to alter the activity of proteins for a much longer period of time (2-4 h) than is typical for phosphorylation. To understand the role of methylation in altering cellular activity, it is useful to identify the protein targets for methylation. Sariban-Sohraby et al. (40) demonstrated that a 95-kDa protein was methylated in the presence of aldosterone. Because this size is consistent with the size of a glycosolated Na+ channel subunit, some investigators have suggested that the target for methylation is one of the Na+ channel subunits (39). Indeed, in recent work, Rokaw et al. (37) demonstrated in vitro methylation of the beta -subunit of Na+ channels in A6 cells.

However, methylation of proteins that controls activity of proteins usually occurs only at a very restrictive consensus sequence: the so-called "CAAX" box (a cysteine residue followed by two aliphatic residues, followed by any residue at the COOH-terminal end of the protein). None of the ENaC subunit sequences contain any cysteine residues that meet the criteria for methylation. However, there are other possible cellular targets for aldosterone-induced methylation of proteins that could regulate Na+ channels. In previous work, using patch-clamp methods, Becchetti et al. (4) have shown that addition of methyl donors to isolated, cell-free patches of membrane to promote protein methylation increases the activity of Na+ channels. Therefore, the target for methylation, if it is not a channel subunit itself, must be a membrane-associated protein in close proximity to the channels. In addition, the same work by Becchetti et al. and also work by Sariban-Sohraby et al. (40) showed that methylation-induced activation of Na+ channels is augmented by cytosolic GTP. Thus methylation of excised, apical membranes induces Na+ channel activity similar to the activity produced by aldosterone, and the methylation reaction is augmented by G protein stimulation. This is a particularly interesting observation, because in one of the many attempts to identify proteins differentially expressed by aldosterone, Spindler and colleagues (42) found that mRNA for the membrane-associated, small G protein K-Ras2a was increased in the presence of aldosterone. K-Ras2a contains the consensus CAAX sequence for methylation and is known to require both GTP and methylation for activity and membrane association (36, 47, 48). The aim of this report is to confirm that aldosterone activates the enzyme isoprenyl-cysteine methyltransferase and induces an increase in cellular protein methylation, and to demonstrate that Ras protein is a target for aldosterone-induced methylation and that Ras might play a role in methylation-induced stimulation of Na+ transport. This work has been reported as a brief communication (1).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A6 cell culture preparation. A6 cells from American Type Culture Collection (Rockville, MD) in the 68th passage (35) were prepared as previously described (25, 29). Experiments were carried out on passages 70-80. Cells were maintained in plastic tissue culture flasks (Corning, NY) at 26°C in a humidified incubator with 4% CO2 in air. The culture medium was a mixture of Coon's medium F-12 (3 parts) and Leibovitz's medium L-15 (7 parts) modified for amphibian cells with 104 mM NaCl-25 mM NaHCO3, pH 7.4, with a final osmolality of 240 mosmol/kgH2O. Besides these components, 10% (vol/vol) fetal bovine serum (Irvine Scientific), 1% streptomycin, and 0.6% penicillin (Hazleton Biologics) were added. For the purpose of this study, A6 cells were plated at confluent density on collagen-coated CM-permeable filters (Millipore) in six-well plates. This sided preparation forms a polarized monolayer with the apical surface oriented upward and net Na+ transport moving from the apical to basolateral surface. The bathing medium was supplemented with 1.5 µM aldosterone and 10% fetal bovine serum during the growth phase before experiments.

O-COOH-methyltransferase activity. Sets of confluent cells from the same passage were grown on permeable supports in six-well plates as described above. After the cells reached confluent density, the cells were incubated in aldosterone-free and serum-free medium for 3 days. Then one-half of the cells were washed with PBS (pH 7.4) and refed with serum-free medium containing 0.1 or 1.5 µM aldosterone. The remaining cells (aldosterone-free controls) were washed with PBS followed by readdition of aldosterone-free and serum-free medium. Cells were incubated for 4 h followed by short-circuit current measurements with an epithelial volt-ohm meter (World Precision Instrument, Sarasota, FL). Cells were then washed with PBS and homogenized with 50 mM Tris · HCl, 1 mM NaEDTA (pH 8)-containing protease inhibitors, and centrifuged at 3,000 g at 4°C for 10 min. Supernatants were used as the enzyme source. The enzyme activity assay was similar to that described previously (14, 26). In brief, the incubation mixture (50 µl) contained 50 mM Tris · HCl buffer (pH 8), 720 nM [3H]adenosyl-methionine (15 Ci/mmol), 100 µM of the methyltransferase substrate N-acetyl-S-farnesyl-L-cysteine, a blocking concentration of a methyltransferase inhibitor when appropriate [S-trans,trans-farnesylthiosalicylic acid (FTS), 3-deazaadenosine (DZA), or S-adenosylhomocysteine (SAH)], and enzyme-containing supernatant. After incubation for 1 h at 37°C, the reaction was stopped by the addition of 50 µl of 20% TCA, and the reaction mix vortexed for 10 s. The isoprenyl-cysteine methyl esters were separated by the addition of 400 µl of heptane to the reaction mix. A fraction (200 µl) of the organic top layer containing the methyl esters was transferred to a small top-free Eppendorf tube and dried under vacuum. Two hundred microliters of 1 M NaOH were added, and the tube was placed upright in a vial containing a small volume of scintillation fluid. The vials were sealed and incubated at 37°C overnight. The strong base hydrolyzes the methyl esters, releasing methanol vapor that partitions into the hydrophobic scintillant. The methyl esters in the vials were counted by liquid scintillation counting. The specific activity of the enzyme is defined as counts per minute of methyl-3H group transferred per minute per milligram of enzyme protein. Protein concentration was determined as described by Bradford (7).

Protein methylation. Sets of confluent cells from the same passage were incubated in aldosterone-free, serum-free, and methionine-free medium for 3 days as described above. On the last day, the media were replaced with equivalent methionine-free medium containing tracer amounts (0.05 mM) of L-[methyl-3H]methionine (Amersham Life Sciences, Arlington Heights, IL), and the cells were allowed to incubate for 24 h to allow the methyl donor, S-adenosyl-L-methionine, to reach labeling equilibrium. Then one-half of the cells were washed with PBS (pH 7.4) and refed with serum-free and methionine-free medium containing 0.1 or 1.5 µM aldosterone. The remaining cells (aldosterone-free controls) were washed with PBS followed by readdition of aldosterone-free, serum-free, and methionine-free bathing medium. Cells were incubated for 4 h followed by short-circuit current measurements. For protein extraction, cells were washed with PBS and centrifuged at 4,000 g for 10 min at 4°C. The cell pellet was treated with 0.5 ml of RIPA buffer (PBS, Na deoxycholate, SDS) to lyse the cells. After incubation for 2 h at 4°C, the sample was spun at 10,000 g for 30 min at 4°C. The supernatant was retained for further examination. To investigate the overall methylation of proteins, the total protein in samples was determined as described by Bradford (7). Aliquots of supernatants with equivalent protein were placed in top-free Eppendorf tubes containing 1 M NaOH, and the tubes were placed upright into scintillation vials containing a small amount of scintillation cocktail. The vials were sealed and incubated overnight at 37°C, and the base labile counts were determined. To investigate the methylation of individual proteins, aliquots of supernatants from lysate protein samples were either used as such or the apical membranes were enriched as described by Sariban-Sohraby et al. (41) and run on gels in paired lanes. One lane was stained with Coomassie blue, whereas the other lane was carefully cut from the gel and sliced into small segments that were placed into Eppendorf tubes containing 1 M NaOH, and the tubes were treated to extract base labile methyl esters as described above and as has been described by others (6, 37-40, 49). The counts remaining in the gel are a measure of total protein in the gel slice, because [3H]methionine not only leads to incorporation of counts into S-adenosyl-L-methionine but is also incorporated (as methionine) into protein. Thus the base labile counts are a measure of methylation and the nonbase labile counts are a measure of protein.

Isolation of Ras. After methylation as described above, supernatants from lysates were immunoprecipitated with the specific anti-Ras antibody (1 µg/ml H-Ras 259 antibody-agarose conjugate, SC-35 AC; Santa Cruz Biotechnology) and allowed to incubate for 24 h at 4°C, followed by centrifugation at 3,500 g for 5 min at 4°C. The pellets were retained and washed three times with buffer. The proteins were released from the agarose by addition of sample loading buffer (glycerol, mercaptoethanol, SDS, Tris · HCl, pH 6.7, bromphenol blue) and boiled at 95°C for 5 min, followed by centrifugation at 10,000 g for 30 min at 4°C. The proteins in the supernatants were separated by SDS-gel electrophoresis (12%) at pH 6.8 to separate the proteins. The proteins on one gel were detected by staining with Coomassie blue, and the other lane was prepared as described above to measure methylation of proteins in the immunoprecipitate. The Coomassie-stained lane was destained and transferred to nitrocellulose for Western blotting to determine whether methylated protein(s) run at the same location as Ras. Briefly, proteins on gels were transferred to nitrocellulose after standard procedures. The nonspecific binding was blocked using 5% milk in Tween 20 and Tris-buffered saline and incubation for 1 h at 4°C, followed by conjugation with the specific anti-Ras antibody for 2 h at 4°C followed by washing and exposure to secondary antibody (0.1 µg/ml v-H-Ras Ab-1 antibody, OP01; Oncogene Research Products, Calbiochem, San Diego, CA). To detect the proteins, the membrane was treated with enhanced chemiluminescence Western blotting analysis following the protocol provided by Amersham (Amersham Life Sciences).

Methylation of Ras. Sets of confluent cells from the same passage were incubated in aldosterone-free and serum-free medium for 3 days. Then one-half of the cells were washed with PBS (pH 7.4) and refed with serum-free medium containing 0.1 µM aldosterone. The remaining cells (aldosterone-free controls) were washed with PBS followed by readdition of aldosterone-free and serum-free medium. Cells were incubated for 4 h followed by short-circuit current measurements with an epithelial volt-ohm meter (World Precision Instrument). Cells were then washed with PBS and homogenized with 50 mM Tris · HCl, 1 mM NaEDTA (pH 8)-containing protease inhibitors and centrifuged at 3,000 g at 4°C for 10 min. Supernatants were used as the enzyme source, and exogenous c-K-Ras (Calbiochem) was added as a target for methylation. After incubation for 1 h at 37°C, Ras was separated from the lysate by immunoprecipitation as described above. To investigate the methylation of Ras, aliquots of immunoprecipitates were run on gels in paired lanes. Ras protein in one lane was detected by Western blotting, whereas methylation of proteins was detected in the other lane by exposure of the dried gel on a phosphoimager.

Antisense inhibition of Ras. To inhibit expression of Ras, we used methods that we have previously used to inhibit expression of another membrane-associated epithelial protein, cystic fibrosis transmembrane conductance regulator (24). Sense and antisense phosphorothioate oligonucleotides corresponding to 21 nt beginning at nt 181 of X. laevis Ras (accession no. AF085280) were synthesized by the Emory University Microchemical Facility. This sequence should inhibit expression of both Xenopus H-Ras and K-Ras2a. A search of GenBank revealed no similarity of this 21-mer with sequences other than Ras. On the 5th to 10th day after plating, selected A6 cells were loaded with Ras sense or antisense oligonucleotides. Cellular uptake was enhanced with the use of a lipid transfection agent (Lipofectamine, Life Technologies), according to the manufacturer's instructions. Briefly, cells were incubated for >4 h with 10 µg/ml Lipofectin containing 10 µM oligonucleotide in serum-free A6 medium added to the basolateral and apical baths, followed by a 2-day incubation in serum-free medium containing 10 µM oligonucleotide and 1 µM aldosterone. For patch clamping, the disks were transferred to the stage of an inverted microscope (Nikon), and experiments were performed as described above. In our previous work, we demonstrated in antisense-treated cells by Western blotting and immunocytochemical localization that p21-Ras protein was reduced to levels 5- to 20-fold below that found in sense-treated or untreated cells (47).

Statistics. Tests for significance were performed using a t-test or one-way ANOVA with Student-Newman-Keuls posttests. Results were considered significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Aldosterone and methylation. To confirm the previously described effect of aldosterone on total protein methylation (38), two batches of six-transwell plates with confluent A6 cells were treated as described in Protein methylation. Figure 1 shows that in three separate experiments, aldosterone significantly increases protein methylation ~2.5-fold over aldosterone-free cells (from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein; P < 0.001, n = 3). On the basis of these data, we decided to measure the activity of the enzyme responsible for this methylation. After having reached confluence for 2 days, the control and test samples consisting of six-transwell plates each were deprived of aldosterone. Then aldosterone was added to the test samples 4 h before extraction. The enzyme activity was determined as described in O-COOH-methyltransferase activity. Figure 2 shows that in three separate experiments, methylase activity was significantly increased from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in the aldosterone-treated cells (P = 0.003, n = 3). These observations suggest that methylation might be involved in aldosterone-mediated epithelial Na+ transport. To examine the characteristics of this methylation, we used three known inhibitors of methyltransferases: FTS, SAH, and DZA. SAH is an end-product inhibitor; DZA causes an increase in SAH levels; and FTS is a strong, competitive inhibitor of isoprenyl-cysteine binding. Of the three, under our experimental conditions, we would expect, as we observed, that FTS should be the most effective blocker, although all three blockers reduced aldosterone-induced methylase activity (Fig. 3). Protein methylase activity significantly increased from 16.1 ± 1.40 in aldosterone-depleted cells to 43.5 ± 4.80 methyl ester cpm/µg protein per hour in the aldosterone-treated cells (P < 0.05, n = 3). In cells treated with aldosterone and 100 µM FTS for 4 h, methylase activity was dramatically reduced to 0.75 ± 0.21 methyl ester cpm/µg protein per hour (significantly less than all other treatments; P < 0.05; n = 3). One hundred micromolar SAH and 300 µM DZA treatment for 4 h reduced methylase activity to 20.0 ± 0.34 and 22.51 ± 1.50 methyl ester cpm/µg protein per hour, respectively (both were significantly different from aldosterone-treated cells, but not different from untreated cells; P < 0.05, n = 3). To examine whether inhibition of methyltransferase activity could alter Na+ transport, we examined the effect of the most effective methyltransferase inhibitor, FTS, on short-circuit current, a direct measure of Na+ flux in A6 cells (15). The medium of two six-well plates was replaced 4 h before the short-circuit current was measured in the samples. Three wells each received, respectively, medium, medium containing 100 µM FTS, medium containing 0.1 µM aldosterone, and medium containing 100 µM FTS, medium, and 0.1 µM aldosterone. Figure 4A shows that, as expected in three separate experiments, application of aldosterone increases short-circuit current >13-fold (from 0.56 ± 0.06 to 7.33 ± 0.49 µA/cm2, significantly different from all other treatments; P < 0.05). Application of FTS reduces short-circuit current in the absence of aldosterone (to -1.07 ± 0.33), but completely blocks any aldosterone-induced increase in short-circuit current (after FTS and aldosterone current is -0.94 ± 0.37 µA/cm2, significantly different from untreated and aldosterone-treated cells; P < 0.05; but not different from FTS treatment in the absence of aldosterone). The effect of FTS is not to disrupt the integrity of the A6 monolayer (despite the negative short-circuit current), because FTS does not alter the resistance of monolayer (Fig. 4B), although aldosterone (in the presence or absence of FTS) does produce a small increase in resistance. The effect of FTS suggests a relationship between isoprenyl-cysteine methylation and the aldosterone-induced activity of amiloride-blockable Na+ channels.


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Fig. 1.   Aldosterone alters protein methylation in A6 cells. In 3 experiments with 2 groups of 6 transwells in each experiment containing confluent A6 cells, 1 group treated with aldosterone-free medium (-aldosterone) and the other group in aldosterone-containing medium (+aldosterone), the extent of methylation was significantly higher in the aldosterone-treated cells (from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein, P < 0.001, n = 3).



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Fig. 2.   Aldosterone stimulates protein methylation by increasing protein methylase activity in A6 cells. In 3 experiments consisting of 2 groups of 6 transwells containing confluent A6 cells, 1 treated with aldosterone-free medium (-aldosterone) and the other in aldosterone-containing medium (+aldosterone), protein methylase activity increased significantly in the aldosterone-treated cells (from 14.0 ± 0.64 to 31.8 ± 2.60 methyl ester cpm/µg protein per hour, P < 0.003, n = 3).



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Fig. 3.   Methyltransferase inhibitors reduce the aldosterone-induced increase in methylase activity. To examine the characteristics of this methylation, we used 3 known inhibitors of methyltransferases: S-trans,trans-farnesylthiosalicylic acid (FTS), S-adenosylhomocysteine (SAH), and 3-deazaadenosine (DZA). Under our experimental conditions with 6 transwells for each condition, the direct blocker, FTS, was the most effective blocker, although all 3 inhibitors reduced aldosterone-induced methylase activity. Protein methylase activity increased significantly from 16.1 ± 1.40 in aldosterone-depleted cells to 43.5 ± 4.80 methyl ester cpm/µg protein per hour in the aldosterone-treated cells (P < 0.05, n = 3). In cells treated with aldosterone and 100 µM FTS, methylase activity was dramatically reduced to 0.75 ± 0.21 methyl ester cpm/µg protein per hour (significantly less than all other treatments; P < 0.05; n = 3). One hundred micromolar SAH and 300 µM DZA reduced methylase activity to 20.0 ± 0.34 and 22.51 ± 1.50 methyl ester cpm/µg protein per hour, respectively (both were significantly different from aldosterone-treated cells, but not different from untreated cells; P < 0.05, n = 3).



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Fig. 4.   The methyltransferase-specific inhibitor FTS reduces short-circuit current but not resistance in aldosterone-free and aldosterone-treated A6 cells. Four groups of cells (3 wells each) in 3 separate experiments were used: 1 group treated with aldosterone-free medium (-aldosterone), a second treated with aldosterone-free media containing 100 µM FTS (-aldosterone + FTS), a third treated with 0.1 µM aldosterone medium (+aldosterone), and a fourth treated with 100 µM FTS + 0.1 µM aldosterone (+aldosterone + FTS). A: as expected, application of aldosterone increased short-circuit current >13-fold (from 0.56 ± 0.06 to 7.33 ± 0.49 µA/cm2, significantly different from all other treatments; P < 0.05). Application of FTS reduced short-circuit current in the absence of aldosterone (to -1.07 ± 0.33), but completely blocked any aldosterone-induced increase in short-circuit current (after FTS and aldosterone, current was -0.94 ± 0.37 µA/cm2, significantly different from untreated and aldosterone-treated cells; P < 0.05, but not different from FTS treatment in the absence of aldosterone). The effect of FTS is not to disrupt the integrity of the A6 monolayer (despite the negative short-circuit current), because FTS does not alter the resistance of monolayer (B), although aldosterone (in the presence or absence of FTS) does produce a small increase in resistance.

Targets for protein methylation. On the basis of the above observations, we decided to investigate possible target proteins for methylation in A6 cells. Two sets of two six-well plates of cells from the same passage were treated to label-methylated proteins (as described in Protein methylation), total cellular protein was extracted, and the methylated proteins were resolved on a gel and quantitated by slicing the gel into molecular weight regions. The base labile counts are a measure of the extent of cysteine methyl esterification (Table 1). The ratio of the base labile counts from the aldosterone-treated vs. untreated cells is a measure of the aldosterone-induced methylation. Figure 5 and Table 1 show in three separate experiments that there are two prominent peaks of methylation, one corresponding to a region near 90 kDa and another in a region around 20 kDa. The high-molecular-weight region may correspond to the 95-kDa methylated protein previously described by Sariban-Sohraby et al. (40). On the other hand, we thought the low-molecular-weight band might actually be due to methylation of a small molecular weight G protein like Ras because it is known to require methylation for activity and membrane association (36, 48), it requires cytosolic GTP for activation (just like methylation-induced activation of Na+ channels), and Spindler et al. (42) and work by us (47) has shown that at least one membrane-associated small G protein, K-Ras2a, is induced by aldosterone. First, we examined A6 cells for the measurable presence of Ras protein. Figure 6 shows that an antibody that recognizes the G protein binding domain of several small G proteins including K-Ras2a and H-Ras can specifically detect p21ras in a Western blot (A) and that the antibody is capable of immunoprecipitating Ras (B). In each case, the band detected by the antibody could be competed by the antigenic peptide (+ peptide lanes). When the amount of total cellular Ras is examined as the density of bands in Western blots or after immunoprecipitation, there is an effect of aldosterone on the amount of Ras protein. Ras protein increases 1.58 ± 0.22-fold (C; means ± SE, n = 11), which is marginally different from 1 (P = 0.026). However, because the antibody precipitates more than one isoform of Ras, larger changes in one type of Ras (like K-Ras) might be obscured if that type were only a small fraction of total Ras protein. Despite the fact that there was only a 58% increase in total Ras protein, aldosterone might still induce Ras activation by methylation. To test this possibility, we labeled cells to equilibrium with [3H]methionine to tritiate the methyl esters in methylated proteins (as described in Protein methylation), after which total cellular protein was immunoprecipitated with the Ras-specific antibody used in Fig. 6. The methylated proteins in the immunoprecipitate were resolved on a gel and quantitated by slicing the gel into molecular weight regions. The ratio of the base labile counts from the aldosterone-treated vs. untreated cells is a measure of the aldosterone-induced methylation. Figure 7A shows in three separate experiments that there is now only one peak at the expected molecular weight for Ras and that aldosterone produces an 11.9 ± 0.40-fold increase in Ras methylation even though the protein content of the gel slice is not changed dramatically.

                              
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Table 1.   Mean methyl ester counts in gel slices



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Fig. 5.   Aldosterone induces methylation of proteins in 2 molecular weight ranges. Two groups of cells were used, 1 treated with aldosterone-free medium, and 1 treated with aldosterone-containing medium. After protein methyl esters were labeled to equilibrium by overnight incubation with [3H]methionine, proteins from cell lysates were resolved on a gel and quantitated by slicing the gel into molecular weight regions. For 5 separate experiments, the ratio of the base labile counts from the aldosterone-treated vs. untreated cells is shown on the vertical axis and is a measure of the aldosterone-induced methylation. There are 2 prominent peaks of methylation, 1 corresponding to a region near 90 kDa and another in a region around 20 kDa.



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Fig. 6.   Aldosterone increases the amount of Ras protein. A: Western blot analysis of A6 whole cell lysate; (-) were from aldosterone deprived cell lysate, (+) were from 4 h aldosterone-treated cell lysates. Peptides were probed with antibody (Ab) + 20-fold H-Ras control peptide (SC-35P). B: immunoprecipitation (IP) of Ras from A6 cell lysate; (-) and (+) lysates were treated as above. Peptides were probed with antibody + 20-fold H-Ras control peptide (SC-35P). C: mean fold change in density of p21Ras bands from aldosterone-free and aldosterone-treated cell lysates calculated for 11 Western blots in which aldosterone-free and aldosterone-treated cell lysates were run in adjacent lanes and the band densities measured. Ras protein increases 1.58 ± 0.22-fold (means ± SE), which is marginally different from 1 (P = 0.026).



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Fig. 7.   Aldosterone induces Ras methylation. A: in 3 separate experiments, 2 groups of cells were used, 1 treated with aldosterone-free medium and 1 treated with aldosterone-containing medium. After protein methyl esters were labeled to equilibrium by overnight incubation with [3H]methionine, proteins were immunoprecipitated from cell lysates with anti-Ras antibody. The proteins in the immunoprecipitate were resolved on a gel, and the extent of methylation was quantitated by slicing the gel into molecular weight regions. The ratio of the base labile counts from the aldosterone-treated vs. untreated cells is shown on the vertical axis and is a measure of the aldosterone-induced methylation. After the immunoprecipitation, there was only 1 peak at the expected molecular weight for Ras, and aldosterone-induced methylation of the peak had increased 11.93 ± 0.40-fold, even though the protein content of the gel slice was unchanged (errors for most points except for the 20-25 kDa point are smaller than the symbol size). The inset shows the autoradiogram from 1 experiment of cell lysates from aldosterone-treated and aldosterone-free cells to which exogenous 14C-SAM and K-Ras had been added and that were subsequently immunoprecipitated with anti-Ras antibody. This result demonstrates that K-Ras can act as a substrate for methylation and that aldosterone increases the extent of methylation. B: the amount of methylation in anti-Ras total immunoprecipitates from cells grown in the presence and absence of aldosterone. These results demonstrate that aldosterone increases methylation and that the antibody is specific for Ras because coincubation with the Ras antigenic peptide reduces methylation to baseline levels.

Even though we would anticipate that the immunoprecipitation of Ras demonstrates that Ras is specifically methylated in response to aldosterone, we also wanted to show directly that Ras protein could be methylated in aldosterone-treated A6 cells. In two batches of cells confluent for 2 days, the control and test samples were deprived of aldosterone. Then aldosterone was added to the test cells 4 h before cell lysates were prepared from both batches of cells. 14C-S-adenosyl-L-methionine (SAM) and commercially available K-Ras protein was added to the lysates. After a 30-min incubation period, Ras protein was immunoprecipitated as described above, and the immunoprecipitated protein was separated on gels. To verify the efficacy of the immunoprecipitation, some of the protein was transferred to nitrocellulose and blotted with anti-Ras antibody with results similar to those shown in Fig. 6. The remainder of the immunoprecipitated protein was also separated on a gel that was subsequently exposed to a phosphoimager screen to resolve the 14C-methylated proteins. The inset of Fig. 7A shows one such autoradiogram and shows that lysates from aldosterone-treated cells produced significantly more methylated Ras. Figure 7B and Table 2 show a summary of methylase activity when K-Ras is used as a substrate for methyltransferase in cellular lysates. Aldosterone is capable of significantly increasing Ras methylation, and the methylation is specific for Ras because it can be inhibited by coincubation of antibody with K-Ras antigenic peptide during the immunoprecipitation.

                              
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Table 2.   Mean methyl ester counts and protein in Ras immunoprecipitates

Aldosterone increases Ras methylation, and inhibiting cellular protein methylation reduces Na+ transport. To determine if Ras activation was directly related to increases in Na+ channel activity, we inhibited Ras expression by treating the A6 cells with antisense oligonucleotides directed against sequences near the translation start site of Ras. Cells were incubated overnight with either Ras antisense oligonucleotides or sense oligonucleotides (as controls), and both groups of cells were treated with aldosterone for 4 h. Patches were examined on the apical surface of the cells, and Na+ channel activity measured as NPo (the product of the number of channels times the open probability of the channels), and then open probability, Po, was calculated. In cells treated with Ras antisense, Po was significantly decreased (from 0.40 ± 0.03 in sense-treated cells to 0.08 ± 0.01; P < 0.0001, n = 11), showing that inhibition of Ras expression decreased Na+ channel activity (Fig. 8). These observations support the idea that epithelial Na+ transport requires expression of a small G protein that must be methylated to promote Na+ channel activity.


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Fig. 8.   Ras antisense oligonucleotides reduce the activity of Na+ channels. To determine if Ras activation was directly related to increases in Na+ channel activity, we inhibited Ras expression by treating the A6 cells with antisense oligonucleotide-directed sequences near the translation start site of Ras. Cells were incubated overnight with either Ras antisense oligonucleotides or sense oligonucleotides (as controls), and both groups of cells treated with aldosterone for 4 h. Patches were examined on the apical surface of the cells. Two representative single channel records from a sense-treated cell (left) and an antisense-treated cell (right) are shown (top). Na+ channel activity measured as NPo, and then Po was calculated (bottom). In cells treated with Ras antisense, Po was significantly decreased (from 0.40 ± 0.03 in sense-treated cells to 0.08 ± 0.01; P < 0.0001, n = 11), showing that inhibition of Ras expression decreased Na+ channel activity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

All of the available biochemical evidence suggests that the number of Na+ channel proteins in the apical membrane of A6 cells does not change in the initial period after aldosterone treatment (13, 22, 23, 31, 34). Our laboratory, using single channel measurements, has demonstrated that aldosterone increases Na+ transport in A6 epithelia by increasing the open time and open probability of the channels (20), possibly through a posttranslational modification of the channel (38, 49). One possible aldosterone-induced modification is methylation of membrane protein and lipid (38). In this scenario, intracellular methyltransferases induced by aldosterone would be responsible for the methylation and modulation of Na+ channel proteins. Indeed, our laboratory, using patch-clamp methods, has shown that methylation occurs in isolated, cell-free patches of membrane (4) and that methylation-induced activation of Na+ channels is augmented by cytosolic GTP, as others have also demonstrated (40). This indicates that the target for methylation must be a membrane-associated protein in close proximity to the channel. If alterations in methylation are responsible for aldosterone-induced increases in Na+ transport, then aldosterone must alter methyltransferase activity. Our data (Fig. 1) show that aldosterone does increase total protein methylation by activating methyltransferase activity (Fig. 2). These two observations confirm the idea that aldosterone alters protein methylation by, in some way, regulating methyltransferase activity. One way of regulating active methyltransferases is by controlling the cytosolic concentrations of endogenous inhibitors. The most common inhibitor is the metabolic end product of methylation, SAH. This is the product that is produced when the endogenous methyl donor, SAM, sometimes referred to as SAMe or AdoMet, transfers its methyl group to a target protein. SAH is a potent inhibitor for all methyltransferases, although the half-maximal inhibitory concentration does vary among different transferases (10, 21). The inhibitory action of SAH or DZA, which prevents the hydrolysis of SAH (52), is obvious in Fig. 3. In other work, we have demonstrated that one aldosterone-mediated regulatory mechanism for methyltransferases is by regulating cellular levels of SAH (45). Figure 3 also shows that a potent prenylated protein methyltransferase inhibitor, FTS (28), will inhibit methylase activity, and that the same inhibitor dramatically reduced aldosterone-stimulated Na+ transport (measured as short-circuit current; Fig. 4). These data suggest that aldosterone modulates Na+ channel activity in A6 epithelia by altering isoprenyl-cysteine methylation. This conclusion is similar to that reached by others using somewhat different approaches (6, 39, 47).

However, on the basis of the results described above and the observation that at least one Ras gene, K-Ras2a, is an aldosterone-inducible gene (42, 47), we thought that one potential target that was known to be activated by both methylation and GTP was a small G protein like Ras. Therefore, we examined the effect of aldosterone treatment on methylation of Ras protein. Figure 5 shows that in the molecular weight range expected for Ras, there is a significant increase in methylation even though the protein content of the gel slice is altered very little. Although there is no significant change in the total protein within the gel slice that includes Ras's molecular weight range, there still might be a change in the amount of Ras protein if Ras makes up only a small fraction of total protein. Figure 6 shows that, in fact, there is a significant change in the total cellular amount of Ras protein. One additional caveat is that the antibody used to detect Ras will detect most cellular forms of Ras. Thus if one type of Ras (like H-Ras) was present at high concentration, then it might be difficult to demonstrate a larger change in a rarer species (like K-Ras). The question remained of whether the methylated protein in the 20-kDa gel slice is actually Ras instead of some other low-molecular-weight protein. In Fig. 7, we show that Ras is a substrate for methylation (Fig. 7B), that aldosterone produces a large increase in the methylation of Ras protein, and that methylated Ras is detectable in autoradiograms and is increased after aldosterone treatment. This demonstrates that although there might be some other low-molecular-weight protein that is also methylated, Ras is strongly methylated in response to aldosterone. Finally, we found it interesting that Ras is methylated in response to aldosterone, whereas others have shown that methylation activates Ras (36, 48); but the question remained of whether Ras has anything to do with Na+ transport. Figure 8 shows that when Ras is inhibited by antisense oligonucleotides, there is a significant reduction in channel activity.

All of these results demonstrate that methylation of Ras is necessary for aldosterone-induced increases in Na+ transport, but methylation alone may not be sufficient by itself to increase Na+ transport. Rather, we feel that Ras methylation is permissive. As we and others have pointed out in other work, neither of the two main enzymes associated with methylation, methyltransferase, or SAH hydrolase, are proteins in which cellular concentration is increased by aldosterone; that is, aldosterone does not increase transcription and translation of either protein (1, 46, 47). Nonetheless, activity of both proteins increases (possibly due to posttranslational phosphorylation of methyltransferase). On the other hand, at least one Ras protein, K-Ras2a, is apparently an aldosterone-induced gene (42, 47), and our results also suggest that some Ras proteins are induced by aldosterone. However, to be active, K-Ras2a must be methylated (36). Therefore, if the magnitude of the aldosterone-induced increase in Na+ transport is to be proportional to the extent of K-Ras2a transcription and translation, then aldosterone must alter methyltransferase activity to ensure that the rate of Ras methylation does not become the rate-limiting step in the induction of ENaC. As mentioned above, this increase in methyltransferase activity is due to activation of preexisting methyltransferase, not to transcription and translation of new methyltransferase.

We have attempted in our work to limit our examination to Ras methylation and its effect on channel activity. However, our results do not preclude the possibility of alternative types of methylation or methylation of alternative proteins; Ras may not be the only protein that is methylated by aldosterone. After all, Fig. 5 shows that there is also a significant increase in methylation in the 80- to 100-kDa range. This range is consistent with previous reports of aldosterone-induced methylation of a 95-kDa protein and methylation of the beta -subunit of ENaC (37, 39, 40). Methylation of other proteins besides Ras would be consistent with a general aldosterone-induced increase in cellular methyltransferase activity. Under such conditions, it would not be surprising that beta -ENaC is methylated, as reported by Rokaw et al. (37). However, the real issue is what sort of methylation is occurring and whether methylated ENaC by itself in the apical membrane of A6 cells is more active than nonmethylated ENaC. In our experiments, we tried to focus on isoprenyl-cysteine methyltransferase, the methyltransferase responsible for cysteine methylation at the consensus CAAX sequence and which is known to methylate Ras proteins. There are no CAAX sequences in any of the ENaC subunits, and, therefore, the subunits cannot be the target of isoprenyl-cysteine methyltransferase. On the other hand, there are other known methyltransferases, but few are known to directly activate proteins. One that does modifies a COOH-terminal leucine (8, 50), but beta -ENaC contains no such residue (gamma -ENaC does). Alternatively, many proteins are methylated on basic and acidic residues. beta -ENaC contains many such residues (but alpha  and gamma  do, also). In eukaryotes, methylation of acidic or basic residues tends to mark proteins for degradation. Methylation of the beta -subunit in this context would be intriguing because some investigators have suggested that turnover and degradation of beta -ENaC regulates the surface expression of functional channels (43, 44). This does not, however, explain why beta -ENaC appears to be activated in lipid bilayers by methylation donors (like SAM) to increase the open probability of Na+ channels (at least ones treated with dithiothreitol) (18). The difficulty with these experiments is that the channels are reconstituted from oocyte proteins (that may contain other proteins that are targets for methylation, but that must necessarily associate with beta -ENaC), and methylation enzymes are found in a total cell cytosolic lysate. Therefore, the oocyte lysate may contain many methyltransferases other than isoprenyl-cysteine methyltransferase that could methylate beta -ENaC or associated regulatory proteins.

An alternative possibility is that Ras protein forms a complex with one of the ENaC subunits as part of the activation process, and the higher molecular weight band represents methylation of Ras in such a complex. The question of Ras-ENaC association underscores the question of the mechanism by which Ras activation induces channel activity. There are several reports of activation of ion channels by Ras protein (11, 12, 17, 27, 30, 32, 51). In most of these reports, some intracellular factor (such as the cytosolic tyrosine kinase, Src, or the Ras GTPase activating protein, Ras GAP) is necessary to support Ras activity. For these mediators, it seems unlikely that there would be Ras activity in excised, inside-out patches as we have previously reported (4). However, there is at least one report of Ras activity in excised patches in which Ras is still capable of activating a nonselective cation channel in mesangial cells (27). In this case, channel activation and Ras activity could be inhibited by application of an anti-Ras antibody to the cytosolic surface of the excised patch. Thus it would appear that all the necessary components for Ras activation of ion channels can be associated with isolated membranes.

We, therefore, conclude that 1) protein methylation is essential for aldosterone-induced increases in Na+ transport; 2) one of the targets for methylation is p21ras; and 3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity. A schematic diagram of the components of aldosterone-signaling cascade described in this paper is shown in Fig. 9.


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Fig. 9.   A schematic diagram of some of the possible components of the aldosterone-signaling cascade depicting the involvement of Ras and methylation. SAM, S-adenosyl-L-methionine.


    ACKNOWLEDGEMENTS

The authors thank B. J. Duke for exceptional patience and good humor in maintaining the A6 cell cultures.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Fellowship F32-DK-09729 (to J. D. Stockand) and Grant R01-DK-37963 (to D. C. Eaton).

Address for reprint requests and other correspondence: D. C. Eaton, Center for Cell and Molecular Signaling, Dept. of Physiology, Emory Univ. School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322.

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. §1734 solely to indicate this fact.

Received 29 July 1999; accepted in final form 21 February 2000.


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DISCUSSION
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