1 Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284; and 2 Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322
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ABSTRACT |
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The A6 cell line was used to study the role of S-adenosyl-L-homocysteine hydrolase (SAHHase) in the aldosterone-induced activation of the epithelial Na+ channel (ENaC). Because aldosterone increases methylation of several different molecules, and because this methylation is associated with increased Na+ reabsorption, we tested the hypothesis that aldosterone increases the expression and activity of SAHHase protein. The rationale for this work is that general methylation may be promoted by activation of SAHHase, the only enzyme known to metabolize SAH, a potent end-product inhibitor of methylation. Although aldosterone increased SAHHase activity, steroid did not affect SAHHase expression. Antisense SAHHase oligonucleotide decreased SAHHase expression and activity. Moreover, this oligonucleotide, as well as a pharmacological inhibitor of SAHHase, decreased aldosterone-induced activity of ENaC via a decrease in ENaC open probability. The kinetics of ENaC in cells treated with antisense plus aldosterone were similar to those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate from open states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gating mode with short open and closed times to one with longer open and closed times.
epithelial sodium channel; hypertension; methylation; S-adenosyl-L-methionine; kidney
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INTRODUCTION |
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ALDOSTERONE IS THE PRIMARY HORMONE modulating Na+ homeostasis. This hormone targets principal cells of the renal collecting duct to increase electrogenic Na+ reabsorption. This discretionary Na+ reabsorption establishes a significant osmotic force favoring dependent water reabsorption. Because blood pressure in humans is, in part, determined by plasma volume, aldosterone-mediated reabsorption is an important modulator of blood pressure. Thus it is important to understand the actions of aldosterone at both a systemic and cellular level. While the systemic actions of aldosterone are well established, namely, plasma volume expansion due to increased Na+ and dependent water reabsorption, the cellular mechanisms of action are enigmatic.
Na+ reabsorption is a two-step process with active transport across the serosal plasma membrane promoting restrictive diffusion of Na+ across the luminal plasma membrane. The activity of the luminal cell-entry pathway is rate limiting and increases in response to aldosterone. Serosal Na+-K+-ATPases maintain the electrochemical gradient for Na+ cell entry across the luminal membrane. Luminal entry is through the amiloride-sensitive Na+-selective ion channel known as epithelial Na+ channel (ENaC). This 4-pS, amiloride-sensitive Na+ channel is well characterized (for reviews, see Refs. 5, 11, and 12).
A critical aldosterone-sensitive methylation reaction is thought to be important to steroid-regulated Na+ reabsorption in some epithelia (24, 29). Aldosterone increases methylation of both lipid and protein through substrate-specific methyltransferases. Transferases transfer a methyl group to a specific protein or lipid target from the methyl donor S-adenosyl-methionine. S-adenosyl-L-homocysteine (SAH) is one end-product of this reaction; the other is methylated substrate. SAH is a potent negative-feedback inhibitor of most methyltransferases. Thus the enzymes responsible for setting cellular SAH level could play an important role in mediating aldosterone-dependent changes in ENaC activity. Considering this, we suggest that the collective activities of the diverse methyltransferases must depend on the activity of SAH hydrolase (SAHHase; EC 3.3.1.1), the only known enzyme in eukaryotes capable of catabolizing SAH (32). An increase in methyltransferase activities independent of a parallel change in SAHHase activity would lead to cellular accumulation of SAH and subsequent end-product inhibition of further methylation. Moreover, stimulation of SAHHase activity would promote methylation by relieving feedback inhibition, which would not distinguish between transferase types, and could explain the general increase in substrate methylation observed in epithelia upon treatment with aldosterone.
We have previously shown that aldosterone induces SAHHase activity, causing a concomitant increase in substrate methylation (27). While increases in SAHHase activity and methylation are well correlated with increased Na+ reabsorption, our understanding of the exact effects of SAHHase on the luminal entry pathway, ENaC, are rudimentary. Moreover, it is unclear whether aldosterone increases SAHHase activity by increasing SAHHase protein or by increasing the activity of existing SAHHase protein. The current research demonstrates that aldosterone increases SAHHase activity independent of enzyme expression. Importantly, we show for the first time that active SAHHase is necessary for stabilization of ENaC in the open state (and also in the closed state) with the end result being that the open probability of ENaC increases. This stabilization facilitates Na+ reabsorption by increasing luminal entry of Na+. Our results are consistent with ENaC being one final effector of aldosterone-induced SAHHase activity.
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MATERIALS AND METHODS |
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Cell Culture
The amphibian renal epithelial A6 cell line (American Type Culture Collection) was used for all experiments (passages 71-80). Cells were maintained in tissue culture by using standard methods described previously (16, 29, 31). Complete but not basic medium was supplemented with 10% (vol/vol) fetal bovine serum and 1.5 µM aldosterone.For all experiments assessing Na+ transport, SAHHase activity, and SAHHase protein levels, cells were plated (~1.0 × 106 cells/cm2) on permeable, 3.8-cm2 tissue culture inserts (0.02 µM Anopore membrane; Nalge NUNC International) and grown to confluence (~2 wk) in complete medium. This enabled cells to establish polarity and form tight monolayers capable of steroid-sensitive vectorial transport. With these conditions, A6 cells avidly transported Na+ from luminal to serosal fluids.
For patch-clamp analysis, cells were plated at confluent density on glutaraldehyde-fixed, rat-tail collagen-coated Millipore-CM filters attached to cover the openings (~5-10 mm2) in the bottom of concave polycarbonate rings (for more detail, refer to Refs. 14, 18, and 20). For at least a week before experimentation, cells were maintained in complete medium to enable this preparation to form a tight, polar epithelium capable of vectorial transport.
To facilitate quantitation of induced Na+ transport and
ENaC activity, we treated confluent monolayers with basic medium for 2 days before experimentation. This treatment established transport at a
basal level. All cellular responses to aldosterone were then easily
normalized. The current study focused on the aldosterone-signal transduction that culminates in activation of preexisting luminal Na+ channels (refer to Refs. 5 and 33). Thus
all experiments unless indicated otherwise were performed 4 h
after addition of aldosterone (1.5 µM) or vehicle (0.1% DMSO) to
cells previously maintained in basic medium for at least 48 h.
Molecular Biological Methods
Overexpression of SAHHase. Creation of a eukaryotic expression construct containing full-length Xenopus laevis SAHHase cDNA has been described (27). This construct, pxSAH, in conjunction with Lipofectamine Plus reagents (Life Technologies), was used to create A6 cells enriched with SAHHase.
Oligonucleotide strategy.
SAHHase antisense oligonucleotide
(5'-GGACAGTTTGTCAGACATGGTG-3') was complementary to and spanned the
translation start codon (4 to 18) of SAHHase mRNA, whereas
sense oligonucleotide (5'-CACCATGTCTGACAAACTGTCC-3') was homologous to
the coding strand. In X. laevis, the only sequence with
identity to these oligonucleotides as described by a standard nucleotide-nucleotide BLAST search [National Center for Biotechnology Information (NCBI)] was SAHHase. Use of these
oligonucleotides has been described previously (27). In
brief, cells were treated with oligonucleotide (5-10 µM, in
basic medium) for 24 h before experimentation. Cells then were
treated with aldosterone (1.5 µM) for 4 h in the continued
presence of oligonucleotide.
Protein Chemistry
Assay of SAHHase activity. A continuous spectrophotometric enzyme-linked assay, described previously (27), was used to quantify SAHHase activity. SAH hydrolysis results in the formation of adenosine and homocysteine. Our assays were performed in the presence of saturating adenosine deaminase (ADA) activity. Thus we followed the decay of SAH first to adenosine and ultimately to inosine with SAHHase activity being rate limiting.
For these assays, whole A6 cell lysate was extracted by Dounce homogenization of 106 cells/ml in 30 mM Tris, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (pH = 8.0). After cellular debris was cleared, the resulting supernatant was maintained at 4°C and used within 2 h as the source of SAHHase. Enzyme activity was measured in the hydrolytic direction in the following reaction buffer (in mM): 25 KH2PO4, 2 MgCl2, and 1 EDTA (pH 7.2). Final reaction volumes of 1 ml contained 698 µl of reaction buffer, 100 µl of 1 mM SAH, 200 µl of whole A6 cell lysate (normalized for protein concentration), and 2 µl of ADA (~5 units; type VIII; Sigma). Reactions were initiated with the addition of lysate, and absorbance changes at a wavelength of 265 nm resulting from SAH metabolism to inosine were recorded with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech). The initial, linear rate of change (1-5 min) was used to calculate activity.Anti-xSAHHase antisera. A peptide corresponding to residues 413-430 of X. laevis SAHHase was synthesized with an amino-terminal cysteine (NH2-CKQAKYLGLDKEGPFKPDHKYR-COOH). This peptide was linked to keyhole limpet hemocyanin and used to immunize rabbits (Lofstrand Labs) to create antisera against xSAHHase (AB725). In X. laevis, the only polypeptide with identity to the injected antigen as described by a standard protein-protein BLAST search (NCBI) was SAHHase. Rabbits were boosted every 0.5 mo for 3 mo and then placed on production boosts (once a month) for 3 mo with antisera harvested 3-8 mo after initial immunization. For this antisera, a 1:32,000 titer against the immunizing SAHHase antigen was established by ELISA (not shown).
Extraction and immunoblotting.
Whole cell lysate was extracted, as previously described (28, 29,
31), with detergent (1% Nonidet P-40) in the following buffer
(in mM): 50 Tris · HCl, 76 NaCl, 2 EGTA, and 10% glycerol (pH = 7.4) supplemented with protease inhibitors (1 µM each of PMSF, leupeptin, N-tosyl-L-phenylalanine
chloromethyl ketone, and
N-p-tosyl-L-lysine
chloromethyl ketone). Crude membrane and cytosolic fractions
were prepared by differential centrifugation after cells were harvested
by sonication in 0.25 M sucrose (10 mM HEPES, pH 7.4). Subsequent to
removal of cellular debris, nuclei, and mitochondria, the microsomal
fraction (P100) was separated from the cytosol (S100) by centrifugation
at 100,000 g for 90 min. For whole organ lysates, tissue was
obtained from three female frogs (X. laevis) maintained on
standard diet. Immediately after they were harvested, organs were
washed twice with ice-cold PBS and transferred to ice-cold lysis buffer
(0.4% sodium deoxycholate, 1% Nonidet P-40, 50 mM EGTA, and 10 mM
Tris · HCl, pH 7.4, supplemented with protease inhibitors).
Organ homogenates were centrifuged at 5,000 g for 10 min and
then at 10,000 g for 30 min to remove debris.
Electrophysiology
Transepithelial electrical measurements. Transepithelial Na+ reabsorption is reported as transepithelial current calculated under open-circuit conditions from the transepithelial voltage and resistance (corrected for the resistance of the bare filter support). The majority of transport across A6 cells under our experimental conditions is carried by Na+ with apical entry being limiting. Moreover, all transport is inhibited by the Na+ channel blocker amiloride. The open-circuit condition, which closely mimics a physiological environment, allows for quantitation of Na+ reabsorption with adequate cellular material available for subsequent biochemical manipulations.
Transepithelial potentials (PD) and resistances (R) before and after treatment with aldosterone or vehicle were measured with the use of a Millicell Electrical Resistance System with dual Ag-AgCl pellet electrodes (Millipore). Equivalent short-circuit current (eqIsc) was then calculated by using Ohm's law, PD = eqIsc · R, with current per area reported.Patch-clamp recording and single-channel analysis.
Patch-clamp recording was performed as described previously
(14, 18, 20, 30). All experiments were performed at room temperature with patch-clamp pipettes of ~5 M. Pipette and
bath solutions were physiological amphibian saline containing (in mM) 100 NaCl, 3.4 KCl, 10 HEPES, 1 MgCl2, and 1 CaCl2 (pH = 7.4). Channel activity was measured with
an Axopatch amplifier (Axon Instruments). Current recordings of ENaC
were made with no applied pipette potential after gigaohm seals (>20
G
) were obtained with the patch electrode on the surface of the A6
cell apical plasma membrane (cell attached). Inward currents (pipette
to cytosol) are shown as downward deflections.
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(1) |
Materials
Reagents were purchased from either Sigma or Calbiochem unless indicated otherwise. Electrophoresis reagents, materials, and equipment were from Bio-Rad. The Emory University Microchemical Facility synthesized the sense and antisense phosphorothiate oligonucleotides.Statistical Analysis
Data are presented as means ± SE unless indicated otherwise. Statistical significance for two sets of data was determined with SigmaStat (Jandel Scientific) using Student's t-test. For multiple comparisons, either a one-way ANOVA in conjunction with the Student-Newman-Keuls posttest or, in a few cases, an ANOVA on ranks with a Dunn's posttest was used. For comparison of two proportions or ratios, a z-test with the Yates correction was used. In one case, for multiple proportions, a Chi-square test with Bonferroni's posttest was used. P ![]() |
RESULTS |
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Characterization of Xenopus laevis SAHHase
Figure 1 shows the characterization of anti-SAHHase antisera AB725. Western blot analysis of whole A6 cell lysate with AB725 antisera identified a protein of 47 kDa. In four experiments, with a typical one shown in Fig. 1A, AB725 interaction with the 47-kDa protein was blocked by preabsorption with 0.3 mg/ml antigen, showing this interaction to be specific. Preimmune antisera did not recognize the 47-kDa protein (not shown). Cells transiently transfected with an expression construct containing SAHHase cDNA (pxSAH) had 2.2 ± 0.2-fold (n = 10) more SAHHase expression compared with control transfectants (Fig. 1B). These results are consistent with AB725 antisera being specific for SAHHase protein, which is ~47 kDa under reducing conditions (32).
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Figure 2 shows that SAHHase in A6 cells
is a cytosolic protein. SAHHase protein, identified with AB725, was
13.3-fold (n = 4) enriched in the supernatant (S100)
after differential centrifugation, suggesting that this protein is
cytosolic (Fig. 2A). In contrast, the -subunits of both
ENaC and the Na+-K+-ATPase localized to the
particulate (P100) fraction (Fig. 2B). These latter proteins
are well established as intrinsic membrane proteins resident to the
apical and basolateral membranes, respectively. The findings that
little SAHHase appears in the P100 fraction but that no ENaC or
Na+-K+-ATPase is detectable in the S100
fraction show successful separation of cytosol from particulate.
Aldosterone did not affect localization of SAHHase in A6 cells
(n = 2, not shown).
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The Western blot in Fig. 3 shows the
tissue distribution and relative abundance of SAHHase in X. laevis (n = 3). The blot probed with AB725 (Fig.
3, top) is of whole tissue lysates. The black arrow
(top) marks an immunoreactive protein of ~47 kDa that is competed with antigen (bottom). The straight line notes a
heavier nonspecific protein identified in both the blot probed with
AB725 alone (top) and AB725 preabsorbed with antigen
(bottom). Below the blots is a representation of the optical
density (in arbitrary units) of SAHHase in the respective tissues
(ordinate is log scale). SAHHase was most abundant in liver followed by
oocyte, kidney, and small intestine. Every tissue tested contained
SAHHase, a finding consistent with the idea that SAHHase is a
ubiquitously expressed enzyme (32).
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Aldosterone Increases SAHHase Activity
Figure 4 shows that aldosterone increases SAHHase activity. Figure 4A shows the rate of SAH hydrolysis. Lysate (normalized for protein concentration) from A6 cells treated with aldosterone (1.5 µM for 4 h) had higher SAHHase activity compared with lysate prepared from control (untreated) cells. Also shown in Fig. 4A is the activity of 1 unit (hydrolytic activity = 1 nM SAH/min at pH 7.2, 37°C) of exogenous rabbit erythrocyte SAHHase (Sigma).
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SAHHase activity was determined from the rate of absorbance decay at 265 nm for the first 5 min for both treated and control conditions. The mean SAHHase activity for aldosterone-treated and untreated cells is reported in Fig. 4B. Each lysate was assayed at least twice. Aldosterone significantly increased SAHHase activity 2.4 ± 0.4-fold (n = 4).
Results in Fig. 5 demonstrate that
the amount of SAHHase protein is not increased by aldosterone. Figure
5A shows a typical Western blot (focusing on SAHHase) probed
with AB725. This blot contained whole cell lysate extracted from A6
cells that were serum and steroid starved for 48 h and then
treated for 4 h with basic medium (control) or medium supplemented
with aldosterone (1.5 µM). Four such experiments are
summarized in Fig. 5B. The optical density (in arbitrary
units) of SAHHase in the absence (control) and presence of aldosterone
were 0.95 ± 0.03 and 0.97 ± 0.10, respectively, and were
not significantly different. These results also show that the apparent
size of SAHHase does not change in response to aldosterone as
determined by SDS-PAGE under reducing condition.
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Functional SAHHase is Necessary for Aldosterone-Induced ENaC Activity
To further investigate the effects of SAHHase activity on Na+ transport, we examined the effect of an SAHHase inhibitor, 3-deazadenosine (DZA), on ENaC. First, paired sets of A6 cells were serum and aldosterone starved for 48 h. One set was additionally treated with 300 µM DZA for 2 h. After this, 1.5 µM aldosterone was added to both sets and ENaC activity was randomly sampled. The aldosterone-sensitive ENaC activity of 0.30 ± 0.19 (mean ± SD, n = 49) in cells pretreated with DZA was significantly lower compared with that of 2.90 ± 0.73 (n = 37) measured in cells treated with aldosterone alone. Po was also significantly reduced by the action of DZA, from 0.41 ± 0.06 (n = 37) to 0.08 ± 0.03 (n = 49). In eight patches (4 untreated and 4 DZA treated) likely containing only a single channel, the Po of ENaC in this limited set was somewhat lower than that calculated for ENaC from patches containing larger channel populations, but DZA still significantly reduced Po from 0.21 ± 0.06 to 0.07 ± 0.01. Representative records and the interval histograms for these patches are shown in Fig. 6. The histograms consist of at least two exponential components reflecting two classes of open events (short and long) and two classes of closed events (short and long). Inhibition of SAHHase with DZA, in addition to reducing Po, reduced the residency time in all states, both closed and open, short or long. However, residency in long closed states was favored over residency in long open states. This results in the decrease in Po. Table 1 provides values for the mean durations of the different classes of events and the calculated Po based on the frequency and duration of the different events.
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While DZA inhibition of SAHHase has a profound effect on ENaC kinetics, we worried about the specificity of a pharmacological inhibitor. Thus we used, in addition to DZA, a more specific inhibitor of SAHHase, an oligonucleotide complementary to that area around the translation start codon of SAHHase mRNA (see MATERIALS AND METHODS).
Western blot analysis (Fig. 7,
A and B) and an activity graph (Fig.
7C) show that SAHHase levels and activity are substantially decreased in cells treated with antisense but not sense
SAHHase oligonucleotide (5-10 µM for 24 h). A
typical Western blot probed with AB725 is shown in Fig. 7A.
The summary graph in Fig. 7B shows that the optical density
of SAHHase in the sense group (n = 4) of 4.9 ± 1.7 arbitrary units is significantly greater than that of 1.6 ± 0.5 arbitrary units in the antisense group (n = 7).
Sense oligonucleotide did not itself affect SAHHase levels
(n = 2) compared with untreated cells.
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The graph in Fig. 7C shows that SAHHase antisense
but not sense oligonucleotide also decreases SAHHase activity. The
SAHHase activity of 1.5 ± 0.2 nM · min1 · mg
1 in the
antisense group is significantly less than the 2.5 ± 0.4 nM · min
1 · mg
1 of the
sense group (n = 5). Before assay, cells were treated with aldosterone (1.5 µM) for 4 h. Moreover, the SAHHase
activity observed in lysate prepared from A6 cells treated with sense
oligonucleotide (and aldosterone) was not different from that of
control cells (treated with aldosterone alone). As noted previously
(27), the sense oligonucleotide does not itself affect
SAHHase activity compared with untreated cells.
If SAHHase activity is important for maintaining normal ENaC activity,
then antisense treatment should reduce transepithelial Na+
transport. Figure 8 shows
aldosterone-sensitive transepithelial current remaining after
pretreatment with sense or antisense oligonucleotide. In the absence of
aldosterone, cells treated with either sense (9.6 ± 1.2 µA/cm2) or antisense oligonucleotide (8.9 ± 1.3 µA/cm2, n = 12) had similar currents,
with the current in both groups not differing significantly from that
reported previously in the absence of aldosterone with no
oligonucleotide (27, 29, 31). Addition of aldosterone to
sense-treated cells increased current (to 29.5 ± 1.7 µA/cm2, n = 12) to an extent comparable
to that previously reported for addition of aldosterone alone.
Conversely, addition of aldosterone to antisense-treated cells produced
a significantly smaller increase in current (to 17.4 ± 2.3 µA/cm2, n = 12). Single-channel analysis
(see below) suggests that antisense only affects a fraction of the
cells. Thus the suppression of aldosterone-sensitive current by
antisense may underrepresent the importance of SAHHase to
Na+ reabsorption in this epithelium.
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The finding that antisense only affects a fraction of treated cells has
been reported previously (13, 17) and is further supported
by single-channel measurements from SAHHase sense- and antisense-treated cells in which, in this batch of cells, only 8 of 25 cells clearly responded to antisense oligonucleotide. The typical
single-channel current traces of Fig. 9,
left, show ENaC in cell-attached patches on cells treated
with either sense (top left) or antisense oligonucleotide
(middle and bottom left) for 24 h, in
addition to aldosterone for 4 h. The channels from sense-treated
cells are not significantly different from those in cells treated with
aldosterone alone (Fig. 6 and Table 1). For the group treated with
antisense, patches with two distinct types of channel kinetics were
observed: one type, which we described as nonresponders (Fig. 9,
middle left), was similar to the sense-treated group, and
the other, which we termed responders (bottom left), was
distinct from both the sense-treated and nonresponder groups in that
the openings were very brief. We categorized a patch as a responder if
it had a mean open time <600 ms (>2.7 times smaller than the mean
open time for all sense-treated patches). None of the treatments
produced any significant change in unitary current measured with no
applied potential (0.29 ± 0.01 pA in sense-treated cells,
0.31 ± 0.03 pA in antisense-treated nonresponding cells, and
0.33 ± 0.04 pA in responding cells).
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We examined a total of 109 patches, 50 after treatment with sense oligonucleotide and 59 after treatment with antisense. Of the 50 sense patches, 19 contained at least one ENaC channel, while in the 59 antisense patches, 25 contained at least one ENaC channel. The frequency of observing at least one ENaC in a patch was not significantly different between the sense and antisense groups. ENaC in the 19 patches from sense-treated cells all had mean open times >600 ms and were indistinguishable from those in cells treated with aldosterone alone (Fig. 6 and Table 1; also see Refs. 3 and 20). Conversely, the antisense-treated group consisted of two populations, with 17 nonresponding patches containing ENaC with gating like that of the sense-treated group (mean open time >600 ms) and 8 responding patches containing ENaC with fast gating (mean open time <600 ms). We tested the likelihood of observing such a proportion of differently gating channels in the antisense group when the sense group contained none by using a z-test and found that these two proportions are significantly different (P < 0.001). This finding suggests that antisense SAHHase significantly affected at least 32% of the treated cells. Moreover, an examination of the Po of the three groups indicated no significant difference between the Po of the sense-treated cells (0.23 ± 0.04) and nonresponding antisense-treated cells (0.23 ± 0.03), but the Po of the responding antisense-treated cells (0.06 ± 0.01) is significantly different from that of both sense-treated and nonresponding antisense-treated cells. In addition, the distributions of Po and mean open times of sense-treated and nonresponding antisense-treated cells are normally distributed with overlapping distributions. The distribution of Po and mean open times of responding antisense-treated cells are also normally distributed but do not significantly overlap either the sense or nonresponding antisense distributions. While ENaC in all patches had two open and two closed states, it was interesting that all channels in a given patch appeared to be affected by oligonucleotide in the same fashion. In other words, ENaC with normal and fast gating were never observed in the same patch. We interpret this as indicating that those cells showing marked changes in channel kinetics were more responsive to antisense oligonucleotides. We have described such a phenomenon before (13).
For each group, Fig. 9 also shows typical dwell-time interval
histograms for open and closed events from three to five patches with a
statistical probability of containing a single channel: sense
(top right), nonresponder (middle right), and
responder (bottom right). In all cases, for both closed and
open times and sense- and antisense-treated cells, the interval
histograms consisted of at least two resolvable exponential components,
implying that ENaC had at least four kinetic states, two open and two
closed, which we designated C1 (a short-duration closed state), C2 (a long-duration closed state), O1 (a short-duration open state), and O2
(a long-duration closed state) with mean residency times of
C1,
C2,
O1, and
O2, respectively. Examination of residency times for the
three groups indicated that there was no statistical difference between
the sense and nonresponder residency times but that responder residency
times were significantly different from those for both sense and
nonresponder. Therefore, in subsequent results and discussion, we
pooled sense and nonresponding cells for comparison with responders.
The residency times associated with the four exponential components of
the interval histograms are summarized in Table
2. For every component in the responding cells, treatment with antisense oligonucleotide produced a substantial reduction in mean duration. The proportion of long events (both open
and closed) also decreased significantly in antisense responding cells.
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Despite the fact that the duration of both open and closed events (both
short and long) decreased, the overall contribution of closed events
increased. Thus we conclude that, like DZA, antisense SAHHase decreased aldosterone-sensitive ENaC activity and
Po without significantly changing N.
Table 3 shows a summary graph of the activity of ENaC from the sense plus nonresponder and responder groups.
The NPo of 0.47 ± 0.03 for sense plus
nonresponders was significantly greater than that of 0.08 ± 0.01 for responding cells. The number of channels per patch is not
significantly different among groups; however,
Po of the responding antisense-treated cells
(0.06 ± 0.01, n = 7) is significantly less than
that of sense plus nonresponding cells (0.23 ± 0.03, n = 13).
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DISCUSSION |
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The current study directly assessed the role of SAHHase in the aldosterone-induced activity of the ENaC. Both whole amphibian kidney and A6 cells had significant SAHHase protein expression. While aldosterone increased SAHHase activity, steroid did not affect SAHHase protein expression, suggesting that aldosterone must initiate some posttranslational activation of the enzyme. SAHHase antisense oligonucleotide and the inhibitor DZA were used to decrease SAHHase activity. Both DZA and antisense oligonucleotide treatment had profound effects on ENaC: decreasing NPo, Po, and mean open and closed times. Interestingly, the kinetics of ENaC in cells treated with aldosterone but in which SAHHase was inhibited with DZA or antisense were similar to those of ENaC in the absence of aldosterone, suggesting that SAHHase is important for aldosterone-induced ENaC activity. Our results as a whole support the hypothesis that aldosterone-induced SAHHase stabilizes all the kinetic states of ENaC in such a manner that Po increases with a concomitant increase in Na+ reabsorption.
To understand why the durations of all states of the channel can
increase or decrease by approximately the same amount and still produce
a change in Po requires additional
consideration. For a channel kinetic scheme containing two open states,
O1 and O2, with mean durations of O1 and
O2, and two closed states, C1 and C2, with mean
durations
C1 and
C2, the
Po depends on the mean time in each of the four
states and the frequency with which these states occur. Therefore,
Po is given as
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(2) |
Consider one of the responding cells that had a single channel in a
patch and in which O1 = 140 ms,
O2 = 1,257 ms,
C1 = 563 ms, and
C2 = 1,878 ms. The relative frequencies of the
different types of events, fO1,
fO2, fC1, and
fC2, were 0.31, 0.19, 0.25, and 0.25, respectively. With these values, Po for this
channel calculated from Eq. 1 is 0.32. This value is
somewhat higher than the mean values for all sense and nonresponding
channels because
C2 is somewhat smaller than the mean
value (Table 2). This calculation also illustrates that taking the mean
values in Table 2 is not an appropriate way to calculate
Po but, rather, that the individual values for
each cell should be used and then a mean calculated Po determined (as given in Table 2). The
question remains, why, if the residency times in all state decreases by
approximately the same amount in DZA or antisense responders, should
the Po decrease (rather than stay the same)?
Consider a patch on a responding cell that contained a single channel
with
O1 = 39.8 ms,
O2 = 188 ms,
C1 = 31 ms, and
C2 = 1,427 ms,
all values significantly lower than similar values for untreated or
nonresponding channels. The relative frequency of the different types
of events, fO1, fO2,
fC1, and fC2, were 0.41, 0.09, 0.19, and 0.31, respectively. Po for this
channel as calculated from Eq. 1 is 0.071. Despite the fact
that all the mean durations decreased, the frequency of short openings
increased at the expense of long openings and the frequency of long
closures increased. The combination of more long closures (even though
they are of shorter duration) and fewer long openings is enough to
decrease Po.
Characterization of A6 Cell SAHHase
SAHHase in A6 cells is a cytosolic protein of ~47 KDa under reducing conditions (Figs. 1-4). We believe AB725 to be only the second anti-SAHHase antibody described and the first used to directly show cytosolic localization of SAHHase. The nucleotide sequence of SAHHase predicts a protein of ~47 kDa. Aiyar and Hershfield (1), using 8-azido analogs of adenosine and cAMP as photoaffinity reagents in addition to a monoclonal antibody against SAHHase, also identified human placental SAHHase as an ~47-kDa protein. In addition, several investigators have purified SAHHase to homogeneity by standard chromatographic procedures from various species (32). The holoenzyme under native conditions is 189-200 kDa with four (likely homologous) subunits of 47 kDa.Others have reported (32) that analysis of SAHHase with conditions favoring high resolution reveals two distinct bands of similar density. The apparent doublet at 47 kDa observed in our Western blots of the cytosolic fraction (Fig. 2A) is consistent with this earlier finding. What causes the doublet pattern is unclear but may be related to the binding of the SAHHase cofactor NAD+.
While others have shown SAHHase activity profiles for differing tissues (32), the current study reports tissue distribution of SAHHase using a specific antibody. Figure 3 demonstrates that in X. laevis, liver followed by oocyte, kidney, and small intestine has the highest concentration of SAHHase. This is not an unexpected finding. SAHHase activity in mammals is thought to be highest in liver (32). Similarly, the high level of SAHHase that we found in kidney is consistent with previous reports that this tissue has the ability to metabolize a substantial amount of SAH (15, 32). The results in both Figs. 1 and 3 are consistent with the epithelial cells of the distal nephron having significant SAHHase levels.
Activation of SAHHase by Aldosterone
The results of Fig. 4 showing increased SAHHase activity in response to aldosterone are consistent with previous reports that SAHHase activity is increased by adrenal corticosteroids (7, 27). The results in Fig. 5 demonstrate that while activity increases in response to aldosterone, the levels of SAHHase protein in A6 cells are not affected by the steroid. Similarly to other steroids, aldosterone manifests a cellular response through induction of gene expression. Our results are inconsistent with SAHHase being an aldosterone-induced gene product itself, but they indicate that SAHHase appears to be regulated by one. Research that addresses the specific mechanism of increased SAHHase activity in response to aldosterone is presently being performed. AB725 will be a useful tool in this pursuit.Active SAHHase is Necessary for Aldosterone-Induced Activation of ENaC
SAHHase expression and activity were decreased with antisense SAHHase oligonucleotide (Fig. 7). Activity also was inhibited with DZA (Fig. 6). Two distinct types of ENaC gating were apparent in cells treated with antisense (Fig. 7): one type that had open and closed time constants similar to that of the control sense group (nonresponders), and another type that exhibited very brief openings and closings (responders). Similar to antisense (responders), DZA also resulted in an increase in rapidity of ENaC gating kinetics for all states (Fig. 6). The kinetic parameters and the unitary single-channel current of ENaC in both the sense-treated and nonresponding antisense-treated groups were indistinguishable from the properties of untreated cells (Fig. 6) and similar to values reported previously in A6 cells (2, 3, 14, 18, 20, 34) and in rat renal cortical collecting tubule (CCT) (8, 10, 21). In contrast, the duration of ENaC open and closed events in the responding group was shorter with substantially decreased activity. The kinetic properties of these channels were statistically indistinguishable from those of DZA-treated cells. We have previously used antisense methods to inhibit expression of a variety of proteins (4, 13, 17, 19, 27, 29). In several cases the inhibition of expression is not complete, ranging from extremely effective with undetectable levels of target protein (4) to between 30 and 70% inhibition (13). In the latter case, the inhibition of expression was variable from experiment to experiment. Inhibition of SAHHase expression appears to fall in this variable category. In our single-channel experiments, antisense treatment produced a significant decrease in NPo, Po, and mean open and closed times in 36% of the patches we examined. For the number of patches we examined, the upper and lower 95% confidence limits on this percentage are 50.9 and 21.1%. Thus, even if there was not variability in the efficacy of antisense treatment, it is statistically possible that as many as one-half of the cells in our sample were affected.Our conclusion that antisense affected between one-fifth and one-half
of A6 cells and that the gating kinetics of ENaC in these cells were
not changed in response to aldosterone (as they were in sense-treated
or nonresponding antisense-treated cells) are based on four
observations: 1) antisense decreased Na+
transport (ENaC is the rate-limiting step in the transcellular pathway); 2) in the antisense group, channels with both
normal and short open times were never seen in the same patch;
3) the pharmacological inhibitor of SAHHase, DZA, affected
ENaC gating kinetics in a manner similar to antisense
SAHHase; and 4) the proportional decrease in
SAHHase activity, SAHHase level, Po, and
transepithelial current produced by antisense or DZA was not significantly different. The fractional reduction in each of these values is presented in Table 4. As
expected, DZA produces the largest inhibition, since it affects all
cells and the effect of DZA on transepithelial current and
Po are close to the same (0.20 ± 0.01 vs.
0.17 ± 0.04 of control, respectively). The effect of antisense on
SAHHase activity and transepithelial current is also similar (0.60 ± 0.13 vs. 0.59 ± 0.08 of control, respectively) and the effect
of antisense on the Po of responding cells
(0.28 ± 0.05 of control) is comparable to the change in
transepithelial current and SAHHase activity after we correct for the
fact that only 36% of cells are represented in the responder category
(which would produce a reduction in current to ~0.74 of control). The reduction in apparent amount of SAHHase protein (to 0.33 ± 0.15 of control) is larger than expected compared with other measures but is
not statistically different. Given the large differences in the types
of experiments, the fractional reductions are quite similar.
|
It is possible that the difference between the effect of antisense on transepithelial current and its effect on Po could be due to our underestimating the number of cells that respond to antisense. Our use of a 600-ms cutoff was somewhat arbitrary (based on normal distributions of mean open time and Po). In fact, though there was no statistical difference in the mean values for the sense-treated cells and the nonresponding cells, five of seven of the individual values obtained from patches with apparently only a single channel are less than all values for sense-treated cells. Thus there may be some partial effect of the antisense oligonucleotides that would be expected to reduce the transepithelial current below levels expected from a simple extrapolation of the Po of ENaC in cells we categorized as responders.
As mentioned earlier, all ENaC openings in A6 cells were described by
two types of open and closed events with mean open time constants
O1 and
O2 and
C1 and
C2 (Figs. 7 and 9). Kemendy et al. (14) in
A6 cells and Palmer and Frindt (22) in CCT principal cells
also showed that ENaC has two open and closed states. All of our
findings are consistent with aldosterone-activated SAHHase stabilizing
all of these states. Antisense SAHHase reduced the open time
constants of ENaC, leading to the appearance of the characteristic
gating associated with the responding cell type, but antisense
oligonucleotide did not affect single-channel unitary current. These
results demonstrate that while SAHHase is important to ENaC gating,
this enzyme does not play a role in determining ENaC conductance.
Because antisense failed to affect unitary conductance, the decrease in
both
O1 and
O2 relative to changes in
C1 and
C2 in response to antisense must
produce, in a large part, the decrease in ENaC activity shown in Figs. 8 and 11.
The open time constants for ENaC for sense-treated and nonresponding cells are consistent with those previously reported for ENaC in A6 cells treated with aldosterone for 6 h (14, 28, 31). In this earlier report, both the longer and shorter open time constants were greater in the presence of aldosterone compared with those in the absence of steroid. In fact, the longer open time constant was 30-fold lower in the absence of aldosterone. The results of the present study and this earlier finding are consistent with the hypothesis that aldosterone via activation of SAHHase stabilizes ENaC open states, resulting in increased Po.
Interestingly, Palmer and Frindt (22) have reported three
types of gating kinetics resulting in distinct
Po for ENaC from rat CCT: 1) low
Po (O1 = 24 ms and
O2 = 123 ms), 2) intermediate Po (
O1 = 95 ms and
O2 = 761 ms), and 3) high
Po (
O1 = 82 ms and
O2 = 1,810 ms). In this study, the authors
suggested that these ENaCs are identical in structure but are merely in
different gating modes. This raises the question, what determines the
gating mode of ENaCs? Our results suggest that SAHHase activity, in
part, plays a role in controlling ENaC gating.
Open times of the low-Po channel described by Palmer and Frindt (22) are similar to those of ENaC in antisense-responding or DZA-treated cells in the present study, and the open times of the high-Po channel are similar to those for channels in our sense-treated and nonresponding cells. We believe both studies are consistent with the idea that aldosterone (possibly via SAHHase) plays a role in ENaC gating. Palmer and Frindt used whole animal manipulations (a low-Na+ diet) to produce high levels of circulating aldosterone. All three types of ENaC gating kinetics thus were observed in animals with high aldosterone levels. Because patches containing only single channels were analyzed in Palmer and Frindt's study, it was unclear whether ENaC with distinct gating kinetics coexisted in the same cell. We have never observed and are not aware of any other study showing multiple types of gating kinetics for ENaCs within the same patch. In the context of our results showing that aldosterone via SAHHase stabilizes ENaC channel states, a possible explanation reconciling Palmer and Frindt's results with ours is that cells containing low Po-type channels simply did not respond to aldosterone. In whole animal manipulations, any one of a number of reasons could result in a set of cells failing to respond to aldosterone. Alternatively, all three of the channel types reported by Palmer and Frindt may have existed in both our sense- and antisense-treated cells and we missed them due to sample size. However, we do not believe this latter explanation to be true because 1) there was a significant difference between the proportion of ENaC with decreased open time constants vs. ENaC with longer open times in the antisense- compared with sense-treated cells; in fact, we never saw the low Po-type channel in the sense-treated cells; 2) in the antisense group, a low Po- and higher Po-type channel were never observed in the same patch; and 3) the decrease in transepithelial Na+ reabsorption across antisense-treated cells is consistent with a change in ENaC activity. Active SAHHase may be a trigger that converts the channels from the low-Po gating mode to that of the higher Po variety.
While our results support the idea that aldosterone-sensitive increases in ENaC activity in amphibian renal epithelia are, at least partially, linked to steroid actions on SAHHase activity, the cellular signaling cascade linking SAHHase activity to changes in ENaC kinetics is not completely clear. However, we and others have shown that the primary effect of SAHHase is to metabolize SAH and, thus, prevent it from becoming a rate-limiting inhibitor of methyltransferases (27, 32). We have also demonstrated that the methyltransferase important for ENaC regulation is an isoprenylcysteine carboxylmethyltransferase (29) that possibly methylates a small G protein, K-Ras2A (2, 31). Aldosterone induces production of K-Ras2A, and subsequent methylation enables K-Ras2A to become active in response to GTP binding (2, 26, 31). Activated K-Ras2A increases the Po of ENaC (3) by an as yet unknown mechanism. On the other hand, while some of the elements downstream of SAHHase are known, how aldosterone increases SAHHase activity also remains to be determined. Because there is no change in SAHHase protein amount, aldosterone likely increases SAHHase activity by promoting posttranslational modification of SAHHase.
Other investigators have been unable to show an effect of methylation on ENaC in rat CCT principal cells (9). This may only mean that methylation is necessary but is not the rate-limiting step for activation of ENaC in rat epithelial cells. Nonetheless, the central importance of this pathway for controlling ENaC in all epithelial tissue capable of Na+ absorption remains unclear. However, in diverse cells, including A6 cells, toad urinary bladder cells and possibly bovine distal collecting duct cells, methylation and, thus, SAHHase appears to play a role in regulating Na+ transport via ENaC (reviewed in Refs. 24 and 29).
The current results describe a novel anti-SAHHase antibody. This is the first time an anti-SAHHase antibody has been used to profile tissue distribution and cellular localization of SAHHase. Our report that SAHHase activity is elevated in A6 cells treated with adrenal steroid is consistent with previously published results (7, 23, 27). While aldosterone enhanced activity, steroid failed to alter SAHHase protein levels, proving that aldosterone does not induce the expression of SAHHase but may lead to posttranslational processing of this protein. Experiments utilizing antisense SAHHase oligonucleotide showed that under conditions where SAHHase protein levels and activity are depressed, a distinct type of ENaC gating characterized by markedly reduced channel open times becomes apparent. Similar results were observed when DZA was used to inhibit SAHHase. The openings and closings for all ENaC in our study could be described by two time constants. This finding suggests that active SAHHase is necessary for stable channel openings and that inhibition of SAHHase blocks the ability of aldosterone to maintain Na+ channels with high open probability. We propose that to increase Na+ reabsorption, aldosterone via SAHHase stabilizes ENaC in all kinetic states but that open states are favored enough to produce an increase in open probability.
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ACKNOWLEDGEMENTS |
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We thank B. J. Duke for excellent technical support.
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FOOTNOTES |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-09729 (to J. D. Stockand) and DK-37963 (to D. C. Eaton).
Address for reprint requests and other correspondence: J. D. Stockand, Dept. of Physiology, Univ. of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-3900 (E-mail: stockand{at}UTHSCSA.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.
Received 2 May 2000; accepted in final form 9 April 2001.
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