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INTRODUCTION |
Endocrine regulation of NaCl and water reabsorption at the
collecting tubule of the kidney is the major site in vertebrates for
maintaining normal blood pressure. Aldosterone is the primary hormone
modulating salt reabsorption across renal epithelial tissue. Aldosterone increases cell entry of Na+ from the urine
across the apical plasma membrane by regulating the activity of the 4 pS, amiloride-sensitive, Na+-selective channel
(ENaC1; see Refs. 1-3).
Similar to other steroids, aldosterone regulates cell activity by
modifying gene expression. Whereas the end result of aldosterone
action, increased Na+ reabsorption, is well known, the
aldosterone-induced proteins and resulting signal transduction pathways
remain poorly described and controversial.
Provocative studies by Sariban-Sohraby and colleagues (4) and Wiesmann
et al. (5) established a correlation between methyl
esterification and acute, aldosterone-induced Na+
transport. Several subsequent studies support further the hypothesis that a critical protein methyl esterification, in part, signals the
acute actions of aldosterone (3, 6-8). The substrate of this critical
methylation remains controversial and is an active area of research.
Results from our laboratory suggest that aldosterone increases methyl
esterification of p21ras in renal epithelial cells (9). A study
by Rokaw et al. (10) provides evidence that
ENaC also may
be an important substrate of aldosterone-induced methylation. Although
methylation is a component of aldosterone regulation of Na+
transport, the signal transduction controlling this covalent modification remains to be described.
The enzymes directly regulating substrate methylation are
methyltransferase and methylesterase catalyzing methyl esterification and methyl ester hydrolysis, respectively. Protein methylation is
analogous to protein phosphorylation both being molecular switches controlling protein activity/locale in a reversible manner.
The predominant, intracellular methyl-donating molecule is
S-adenosyl-L-methionine (AdoMet). Methyltransferase
catalyzes the transfer of a methyl moiety from AdoMet onto a substrate
with the end product S-adenosyl-L-homocysteine
(SAH) being formed. This end product is a potent feedback inhibitor of
most transmethylation reactions involving AdoMet as the methyl donor
(11). The observation by Sariban-Sohraby et al. (4) that
aldosterone increases methylation of both protein and lipid is
consistent with the notion that a general regulator of transmethylation
reactions is the control point for mineralocorticoid-induced
methylation. Thus, regulation of cellular SAH concentration may be an
important site for controlling methylation relevant to aldosterone
signal transduction.
The only enzyme known to hydrolyze SAH in vertebrates is
S-adenosyl-L-homocysteine hydrolase (SAHHase; EC
3.3.1.1; see Ref. 11). This enzyme reversibly metabolizes SAH with
hydrolysis resulting in production of adenosine and
L-homocysteine. The cDNA encoding SAHHase has been
identified in various species including Xenopus laevis
(12).
Pharmacological and molecular inhibitors of SAHHase and overexpression
of SAHHase were used to test directly the notion that SAHHase activity
regulates Na+ reabsorption. Since cellular SAH levels as
set by hydrolase activity may regulate Na+ transport
through modulation of substrate methyl esterification, the hypothesis
that aldosterone increases SAHHase activity with concomitant increases
in methylation also was tested. The results of the current study
demonstrate that the natriferic actions of aldosterone are dependent on
SAHHase activity with aldosterone-induced SAH hydrolysis promoting
substrate methyl esterification and subsequent induction of
Na+ reabsorption. Regulation of SAHHase activity by
aldosterone was shown to be one control site for
mineralocorticoid-induced methylation and Na+ reabsorption.
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EXPERIMENTAL PROCEDURES |
Cell Culture
Amphibian distal tubule principal cells (A6 cells; American Type
Culture Collection, Rockville, MD) were maintained in 4% CO2 at 26 °C in a mixture of Coon's F-12 (3 parts) and
Leibovitz's L-15 (7 parts) media (Irving Scientific, Santa Ana, CA)
supplemented with 104 mM NaCl, 0.6% penicillin, 1.0%
streptomycin, 10% (v/v) fetal bovine serum, 25 mM
NaHCO3, and 1.5 µM aldosterone (complete media). Basic media were without aldosterone and fetal bovine serum.
Molecular Biological Methods
Cloning of X. laevis SAHHase--
Single-stranded cDNA from
A6 cells was created using the Marathon cDNA amplification kit
(CLONTECH Laboratories, Inc., Palo Alto, CA) and
poly(A)+ mRNA harvested with the FastTrack 2.0 Kit
(Invitrogen, Carlsbad, CA). An X. laevis SAHHase full-length
clone was amplified from this cDNA using a polymerase chain
reaction in conjunction with specific primers developed from the
reported X. laevis SAHHase sequence (see Ref. 12;
GenBankTM accession number L35559) as follows: forward
primer, 5'-TTCACCATGTCTGACAAACTGTCC-3'; reverse primer,
5'-TGGAGGAAGATTGGTAAAAGAAAGC-3'. Subsequent to agarose gel
electrophoresis (1.2%; TAE) to separate polymerase chain reaction
products, a 1.8-kilobase pair fragment consistent with xSAHHase was
isolated and ligated into pGEM-T Easy (Promega, Madison, WI). The
insert then was subcloned into pcDNA3.1/Zeo(
) (Invitrogen) using
NotI. Nucleotide sequence data from this new expression
plasmid (pxSAHHase3.1(
)zeo) was homologous to that reported by Seery
et al. (12) and was consistent with the plasmid containing
the full-length clone for xSAHHase. As shown in Fig. 1, in vitro translation of
pxSAHHase3.1(
)zeo using the TNT T7 Coupled Reticulocyte Lysate System
(Promega) produced a protein of appropriate size (~48 kDa) for a
complete open reading frame.

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Fig. 1.
Autoradiograph of products separated by
SDS-polyacrylamide gel electrophoresis (10%) from an in
vitro translation of pxSAHHase3.1( )zeo
(left) and negative control (right)
in the presence of [35S]methionine.
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Transfection--
LipofectAMINE PLUS reagents (Life
Technologies, Inc.) were used for transfection of A6 and HEK293 cells.
Transfection followed closely the standard protocol for these reagents
only differing in the fact that for A6 cells incubation time was
increased to 18 h. Transient transfected HEK293 cells were
maintained in tissue culture using standard techniques and used for
experimentation 2-3 days post-transfection. In contrast, A6 cell
transfection was prolonged by zeocin (400 µg/ml) selection.
Subpassages (up to four passages after transfection) of the extended
transfected A6 cells were used for experimentation. Control
transfectants contained either pcDNA3.1/Zeo(
) vector or pVgRXR
(Invitrogen). Neither of these plasmids affected cell activity.
Oligonucleotide Strategy--
Antisense oligonucleotide
(5'-GGACAGTTTGTCAGACATGGTG-3') was complementary to and
spanned the translation start codon (
4 to 18) of xSAHHase mRNA,
whereas sense oligonucleotide (5'-CACCATGTCTGACAAACTGTCC-3') was
homologous to the coding sequence of the same region. In X. laevis, the only sequence with identity to these oligonucleotides as described by a blastn (National Center for Biotechnology
Information) search was SAHHase. Addition of exogenous single-stranded
oligonucleotides to A6 cells followed a protocol similar to that
previously described by this laboratory (13). Briefly, competent A6
cell monolayers washed with phosphate-buffered saline were treated for
24 h prior to experimentation with 5-10 µM
oligonucleotide dissolved in basic media. Aldosterone then was repleted
for 4 h in the presence of oligonucleotide.
Northern Blot Analysis--
For Northern blot analysis, 3-5
µg of poly(A)+ RNA isolated from A6 cells was separated
by electrophoresis on 1.2% agarose/formaldehyde gels, transferred to
nylon membranes, and probed with radiolabeled oligonucleotides of
interest. Probes were produced by nick translation and incorporation of
[32P]dCTP. Northern blots were hybridized and washed
under high stringency conditions and then imaged on a PhosphorImager
after 1-24 h exposure. Band density was quantified using SigmaGel
software (Jandel Scientific).
Assays of Enzyme Activity
SAHHase Activity--
A continuous spectrophotometric,
enzyme-coupled assay similar to that reported by Palmer and Abeles (14)
and Hershfield et al. (15) was used to quantify SAHHase
activity. In brief, whole cell lysate was collected by Dounce
homogenizing cells washed 2 times with phosphate-buffered saline in 1 ml/106 cells of lysis buffer (30 mM Tris, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, pH
8.0). Cellular debris were precipitated by centrifugation at
10,000 × g for 30 min at 4 °C. The resulting
supernatant maintained at 4 °C was used as the source of SAHHase
with enzyme activity being assayed within 2 h after
centrifugation. With the addition of exogenous adenosine deaminase (EC
3.5.4.4), SAHHase activity in whole cell lysate becomes rate-limiting
for the metabolism of SAH to inosine. Enzyme activity was measured in
the hydrolytic direction in the following reaction buffer: (in
mM) 25 KPO4, 2 MgCl2, 1 EDTA, pH
7.2. The final reaction volume was 1 ml with 698 µl of reaction
buffer, 100 µl of 1 mM SAH, 200 µl of A6 whole cell
lysate, and 2 µl of adenosine deaminase (~5 units; Sigma, type
VIII). The reaction was initiated after addition of lysate, and
absorbance changes at
265 resulting from SAH metabolism
to inosine were recorded using an Ultrospec 3000 spectrophotometer (Amersham Pharmacia Biotech). The initial rate of absorbance change (1-5 min) was used to establish activity.
Substrate Methylation--
Transmethylation was quantified by
alkaline hydrolysis of methyl esters in a vapor phase assay similar to
that described by Clarke et al. (16). In brief,
N-acetyl-S-farnesyl-L-cysteine (AFC)
was used as a methyl acceptor, AdoMet as a methyl donor, and A6 whole
cell lysate as the source of methyltransferase. Subsequent to washing 3 times with phosphate-buffered saline, cells were extracted at 4 °C
by Dounce homogenization (1 ml/106 cells) in 50 mM Tris-HCl plus 1 mM phenylmethylsulfonyl
fluoride, pH 8.0. Cellular debris were precipitated by centrifugation
at 10,000 × g for 30 min at 4 °C. Methylation of
AFC was assayed in 50 µl of lysate supplemented with 720 nM [methyl-3H]AdoMet (77 Ci/mmol; Amersham
Pharmacia Biotech) and 100 µM AFC. After incubation for
1 h, the reaction was stopped with 50 µl of 60% trichloroacetic
acid. Methylated substrate was isolated by organic extraction in 400 µl of heptane. The organic phase was subsequently dried and subjected
to base hydrolysis (1 M NaOH). Under these conditions, only
methyl esters (e.g. methylated AFC) are hydrolyzed.
Liberated radioactivity was assayed with a Tri-Carb liquid
scintillation analyzer (model 1900CA; Packard Instrument Co.).
Assay of Cellular AdoMet and SAH Concentrations--
The methyl
donor (AdoMet) and end product (SAH) concentrations were quantified
using standard paper chromatography. Confluent monolayers of A6 cells
were maintained in methionine-free, basic media for 48 h. One day
prior to experimentation, cell media were supplemented with tracer
amounts (50 µM) of [35S]methionine (1157 Ci/mmol; ICN Pharmaceuticals, Inc., Irvine, CA). Aldosterone (1.5 µM) was repleted for 4 h and cells collected in 50 mM Tris-HCl buffer as above. Metabolites were separated by
paper chromatography on Whatman 3MM paper. To locate radiolabeled AdoMet and SAH, 5 µl of a mixture of 1 mM non-labeled
methionine, homocysteine, cysteine, AdoMet, and SAH was run in parallel
with each sample. Metabolite location was identified upon development using ninhydrin reagent plus heat. Two solvents were used in series to
separate metabolites as follows: 1) n-butyl alcohol/glacial acetic acid/water (65:150:20, v/v), and 2) isopropyl
alcohol/ammonia/water (70:10:20, v/v). Subsequent to localization of
metabolites, corresponding spots were removed from the paper, and
radioactivity was quantified using the Tri-Carb liquid scintillation analyzer.
Transepithelial Electrical Measurements
For assessment of transepithelial potentials (PD) and
resistances (R), A6 cells were plated on permeable, 25-mm tissue
culture inserts (0.02 µM Anopore membrane; Nalge NUNC
International, Naperville, IL) and grown to confluency (approximately
10 days) in the presence of complete media. Monolayer competency was
determined by resistance measurement (
1.0 kiloohm/cm2).
Prior to experimentation, competent monolayers were treated with basic
media for 48 h. to decrease Na+ transport to a basal,
non-steroid-induced state. This enabled the dissociation of the acute
from chronic actions of aldosterone. Monolayers then were repleted with
aldosterone (1.5 µM) with or without experimental agents
for 4 h. Transepithelial PD and R before and after treatment were
measured using a Millicell Electrical Resistance System with dual
Ag/AgCl pellet electrodes (Millipore Corp.). Equivalent short circuit
current (eqIsc) was calculated using Ohm's
law: PD = eqIsc × R. Aldosterone-induced
current in this system was amiloride-sensitive and carried by
Na+.
Materials
All reagents were purchased from Sigma or Calbiochem unless
indicated otherwise. Sense and antisense phosphorothioate
oligonucleotides were synthesized by the Emory University Microchemical
Facility. This facility also synthesized the specific X. laevis SAHHase primers used for polymerase chain reaction. For
each lysate, protein concentration was established using the
DC Protein Assay Kit (Bio-Rad).
Statistical Analysis
Data are expressed as the mean ± S.E. Statistical
significance was determined using Student's t test for
paired and unpaired data as appropriate. For multiple comparison, a
one-way analysis of variance in conjunction with the
Student-Newman-Keuls test was used. A p
0.05 was
considered significant.
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RESULTS |
Regulation of Na+ Transport and SAHHase Activity by
3-Deazaadenosine--
Shown in Fig. 2
are the time course and dose-response profiles of the actions of DZA
(4-amino-1-[
-D-ribofuranosyl]-1H-imidazo[4,5]-pyridine) on steady state Na+ transport in A6 cells maintained in
complete media. Maximum current inhibition occurred within 2 h
after application. At 2 h, relative currents were 0.64 ± 0.07, 0.44 ± 0.17, 0.31 ± 0.01, 0.18 ± 0.22, and
0.04 ± 0.02 and were significantly different than currents at
time 0 h for all doses of DZA (30, 50, 100, and 300 µM, respectively; n = 6) and amiloride.
In contrast, relative current at 2 h across monolayers treated
with vehicle (0.95 ± 0.09) was not different compared with 0 h. Moreover, all relative currents at 2 h were different compared
with vehicle at 2 h. The half inhibitory concentration for DZA was
between 40 and 50 µM. Interestingly, current inhibition by DZA at concentrations <100 µM lasted only 4 h,
after which (
6 h) current tended toward levels prior to addition.
Relative current at 8 h for the 30 µM group was
significantly greater than cells treated with vehicle.

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Fig. 2.
Time- and dose-response of DZA inhibition of
steady state Na+ current. Currents at all times are
relative to currents at time 0 h. Concentration of DZA is noted at
the right. Vehicle = 0.3% methanol; amiloride = 5 µM.
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The effects of SAHHase inhibition on aldosterone-induced current and
cellular concentrations of AdoMet and SAH are shown in Fig.
3. The changes in current induced by
aldosterone in the absence and presence of DZA are shown in Fig.
3A and summarized in Table I.
Addition of aldosterone (1.5 µM) for 4 h to
monolayers serum and steroid deprived for 48 h significantly
increased eqIsc by 1.84 ± 0.11 µA/cm2 (n = 16). In the presence of 50, 100., and 300 µM DZA, aldosterone significantly increased
current (
eqIsc = 1.05 ± 0.06, 0.82 ± 0.13 and 0.67 ± 0.13, respectively) compared with
base line; however, induced current in the presence of DZA (at all
doses) was significantly lower compared with that in the absence of
DZA.

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Fig. 3.
Regulation of aldosterone-induced current and
AdoMet (SAM) and SAH catabolism by DZA.
A, repleted is addition of 1.5 µM aldosterone
for 4 h (n = 16); and +50 (n = 18), +100 (n = 18), and +300 (n = 17)
µM are repletion in the presence of DZA (at respective
concentration). Aldosterone repletion in the absence and presence of
DZA increased current above base line. *, p < 0.05 versus repleted control; **, versus +50
µM. B, changes in AdoMet and SAH levels are
relative to aldosterone-depleted cells. AdoMet and SAH levels decreased
approximately 15 and 6%, respectively, in response to aldosterone.
However, in response to aldosterone in addition to 300 µM
DZA (+DZA), AdoMet levels decreased only by about 3% and SAH levels
increased nearly 15%.
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Fig. 3B shows the percent change in AdoMet and SAH levels in
A6 cells in response to aldosterone in the absence and presence of the
SAHHase inhibitor, DZA. AdoMet levels significantly decreased by
15.6 ± 1.7% (n = 6) in response to aldosterone.
Similarly, aldosterone significantly decreased SAH levels by 5.8 ± 1.5% (n = 6). In the presence of DZA (300 µM), aldosterone-induced AdoMet catabolism was
significantly attenuated with levels being only 2.5 ± 0.5%
(n = 6) lower than the concentration prior to steroid addition. Upon simultaneous addition of DZA with aldosterone, SAH level
significantly increased 14.6 ± 0.1% (n = 6),
which is significantly greater compared with that in the absence of
inhibitor. These observations demonstrate that inhibition of SAHHase
activity decreases SAH metabolism resulting in a concomitant decrease
in AdoMet metabolism.
Regulation of methyltransferase activity in A6 cells by end product
inhibition was confirmed. Transmethylation of pseudosubstrate (AFC) in
response to enzymes contained in whole cell lysate was significantly
inhibited by SAH (100 µM) from a control of 43.46 ± 4.80 cpm/µg to 20.04 ± 0.34 cpm/µg (n = 3;
data not shown in a figure).
Fig. 4 shows that aldosterone increases
SAHHase activity in cell lysate but does not increase significantly
xSAHHase mRNA levels. Active SAHHase was inhibited by DZA. As shown
in Fig. 4A (and summarized in Table
II), application of aldosterone for 4 h to serum- and steroid-starved cells significantly increased SAHHase activity from 1.19 ± 0.19 (n = 5) to
2.38 ± 0.26 (n = 8) nM/min/mg.
However, aldosterone-induced SAHHase activity was significantly
attenuated to 0.39 ± 0.10 (n = 8) upon addition of DZA (10 µM) to cell lysate during enzyme assays.
Northern analysis in Fig. 4, B and C, shows that
mRNA identified by a radiolabeled xSAHHase probe does not increase
in response to aldosterone. In such experiments (representative blot
shown in Fig. 4B and 5 experiments summarized in Fig.
4C), the ratio of xSAHHase to GAPDH mRNA did not
significantly change in response to aldosterone application for 4 h with the steroid-depleted and repleted ratios being 1.04 ± 0.30 (n = 3) and 1.58 ± 0.34 (n = 5),
respectively.

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Fig. 4.
Regulation of SAHHase activity
(A) and mRNA (B and
C) by aldosterone. A, depleted cells
( ) were aldosterone- and serum-deprived for 48 h; repleted cells
(+) were subsequently treated with aldosterone for 4 h; and
DZA = assay performed in the presence of 10 µM
inhibitor. *, p < 0.05 versus depleted; **,
versus repleted. Shown in B is a typical Northern
blot. GAPDH and SAHHase are mRNA species identified by specific
probes. C, graphic representation of the relative density of
SAHHase versus GAPDH mRNA in the absence and presence of
aldosterone. Paired experiments from the same blots are indicated by
connecting lines. Relative density for depleted and repleted
were not different (p = 0.33).
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Table II
Regulation of SAHHase activity by aldosterone in knock-out and
overexpressing cells
All values are mean ± S.E. nM/min/mg. DZA = 10 µM, sense and antisense = cells treated with ~5
µM oligonucleotide for 24 h prior to
experimentation; pControl = cells transfected with control
plasmid.
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Antisense Inhibition of Aldosterone-induced SAHHase Activity and
Current--
Application of oligonucleotides complementary to the
region of the xSAHHase sequence around the translation start site
reduced SAHHase activity and aldosterone-induced Na+
transport (Fig. 5). Fig. 5A
(and summarized in Table II) shows that the SAHHase activity in cells
treated with aldosterone for 4 h and sense oligonucleotide
overnight (2.76 ± 0.42 nM/min/mg; n = 7) was not significantly different from the SAHHase activity in cells
treated with steroid alone (2.38 ± 0.26 nM/min/mg;
n = 8; Fig. 4A). However, the SAHHase
activity in cells that were treated with aldosterone for 4 h and
antisense oligonucleotides overnight (1.26 ± 0.21 nM/min/mg; n = 7) was significantly lower than cells treated with aldosterone and sense oligonucleotides or cells
treated with steroid alone.

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Fig. 5.
A, regulation of aldosterone-induced
SAHHase activity by oligonucleotide. Sense and antisense
(ANTI) treated with ~5 µM of the respective
oligonucleotide for 24 h prior to aldosterone repletion. *,
p < 0.05 versus sense. B, the
eqIsc upon treatment of A6 cell monolayers
with basic media (WASH), aldosterone (1.5 µM
for 4 h, ALDO) repletion to control cells, and to those
previously treated with sense and antisense (ANTI)
oligonucleotide are shown. *, p < 0.05 versus wash; **, versus repleted control and
sense.
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Aldosterone-induced current also was inhibited by antisense but not
sense oligonucleotides (Fig. 5B and Table I). In
sense-treated cells, aldosterone significantly increased
eqIsc by 1.6 ± 0.10 µA/cm2
(n = 63), an increase similar to the response of cells
treated with steroid alone. In contrast to sense-treated cells,
pretreatment with antisense oligonucleotides significantly decreased
aldosterone-induced current by approximately 50% to 0.82 ± 0.10 µA/cm2 (n = 107). Note that simply
washing steroid and serum-starved cells with basic media failed to
significantly change current (n = 18).
Overexpression of SAHHase Increased Aldosterone-induced SAHHase
Activity, Methylation, and Na+
Reabsorption--
Overexpression of SAHHase in HEK293 cells increased
hydrolase activity and AFC methylation (Fig.
6). Fig. 6A shows that the hydrolase activity in pxSAHHase3.1(
)zeo-transfected HEK293
cells of 1.66 ± 0.27 nM/min/mg
(n = 6) is significantly greater than the 0.90 ± 0.16 nM/mg/min (n = 6) observed in
control-transfected cells. Moreover, the methylation of AFC, depicted
in Fig. 6B, of 71.4 ± 11.7 cpm/µg × 10
2 (n = 7) in lysate from
pxSAHHase3.1(
)zeo-transfected HEK293 cells is significantly greater
compared with the 39.6 ± 8.3 (n = 9) cpm/µg/min × 10
2 in control transfectants.

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Fig. 6.
Increased SAH hydrolysis (A)
and substrate methylation (B) in transfected HEK293
cells. A, the SAHHase activity (n = 6)
in cells overexpressing SAHHase was greater compared with control
transfectants. B, substrate methylation of AFC was
significantly increased in SAHHase overexpressing cells
(n = 9) compared with control transfectants
(n = 7).
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Transfection with pxSAHHase3.1(
)zeo of A6 cells, shown in Fig.
7, increased both xSAHHase mRNA level
and SAHHase activity. As shown in the representative Northern blot
(Fig. 7A), A6 cells transfected with pxSAHHase3.1(
)zeo had
more xSAHHase mRNA compared with control transfectants. Four such
experiments are summarized in Fig. 7B. The ratio of relative
band densities for xSAHHase to GAPDH is significantly increased
2.65-fold in pxSAHHase3.1(
)zeo versus control (relative
density of xSAHHase/GAPDH = 1) transfectants. Fig. 7C
(summarized in Table II) demonstrates that similar to message level,
SAHHase activity in the absence of aldosterone and serum is increased
in A6 cells transfected with pxSAHHase3.1(
)zeo (2.36 ± 0.37 nM/min/mg; n = 5). Aldosterone addition
significantly increased activity in pxSAHHase3.1(
)zeo
transfectants to 4.91 ± 0.66 nM/min/mg
(n = 8). The activity in response to aldosterone for
SAHHase-overexpressing cells was significantly greater compared with
that for control transfectants (2.82 ± 0.50 nM/min/mg; n = 8). Note that the activity
in aldosterone-treated control transfectants was not different than
that in steroid-treated, non-transfected cells.

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Fig. 7.
Increased SAHHase mRNA (A
and B) and activity (C) in A6
cells overexpressing SAHHase (pxSAH) compared with
control plasmid (pCON). Shown in A is
a typical Northern blot hybridized with probes directed against SAHHase
and GAPDH in experimental (left lane) and control
(right lane) transfectants. Summarized in B
(pairs connected by line) is the increase (*,
p < 0.05) in SAHHase versus GAPDH mRNA
for SAHHase-overexpressing cells (0.90 ± 0.14) in four such
northerns. Relative density is expressed as arbitrary units and for
control transfectants was 0.34 ± 0.02. Graphed in C
are the increases in SAHHase activity upon aldosterone repletion in
pxSAHHase3.1( )zeo (black) and control transfectants
(gray). *, p < 0.05 versus
depleted; **, versus repleted, pxSAHHase3.1( )zeo.
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Overexpression of SAHHase in addition to increasing enzyme activity
potentiated aldosterone-induced eqIsc (Fig.
8 and Table I). Cells overexpressing
SAHHase did not have a significantly elevated basal current compared
with control transfectants and non-transfected cells but did have a
significantly increased current response to aldosterone repletion (1.5 µM; 4 h). Aldosterone significantly increased
current by 1.6 ± 0.13, 1.7 ± 0.06, and 2.6 ± 0.16 µA/cm2 in control-transfected (n = 22; 1st black bar), nontransfected (n = 45; not shown in this figure), and
pxSAHHase3.1(
)zeo-transfected (n = 18; 1st
gray bar) A6 cell monolayers, respectively. The
aldosterone-induced current in SAHHase-overexpressing cells was
significantly greater compared with that in control transfectants and
non-transfected cells. Aldosterone-induced current in all
transfectants was amiloride (5 µM)-sensitive with
inhibitor causing a significant decrease in steroid-increased
Na+ transport. In the presence of aldosterone and amiloride
current in pxSAHHase3.1(
)zeo transfectants decreased from basal
levels by
0.281 ± 0.04 µA/cm2 (2nd
gray box). Currents in the presence of aldosterone after amiloride addition for control and pxSAHHase3.1(
)zeo transfectants were 0.53 ± 0.10 and 0.65 ± 0.06 (n = 12)
µA/cm2, respectively. These currents are not
significantly different to currents in the absence of steroid.

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Fig. 8.
Regulation of current by aldosterone in
transfected A6 cells. Repleted (left) is aldosterone
(1.5 µM; 4 h.) addition to serum- and steroid-
deprived cells. +amiloride (right) is subsequent
addition of 5 µM amiloride to repleted cells.
Black and gray boxes represent
eqIsc from depleted base currents for
control and pxSAHHase3.1( )zeo transfectants, respectively. *,
p < 0.05 versus control transfectants; **,
versus appropriate repleted group.
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A summary of the short circuit current induced by aldosterone repletion
to A6 cells overexpressing SAHHase and in the presence of molecular and
pharmacological SAHHase inhibitors is shown in Table I. Aldosterone
(1.5 µM) addition for 4 h significantly increased
current in control A6 cells, whereas merely changing the bathing medium
without addition of steroid had no effect. Aldosterone also increased
current in transfected cells and cells treated with antisense and DZA.
Basal (depleted) currents for DZA and antisense-treated cells and
transfected cells were not different from appropriate controls. In DZA
and antisense-treated cells, aldosterone-induced current was decreased
compared with control cells and sense-treated cells, respectively.
Aldosterone increased current greater in pxSAHHase3.1(
)zeo
transfectants compared with control cells and control transfectants.
The regulation of SAHHase activity by aldosterone in the presence of
pharmacological and molecular inhibitors of SAHHase and in
overexpressing A6 cells is summarized in Table II. Aldosterone repletion increased activity in control and overexpressing cells; however, the activity in aldosterone-treated
pxSAHHase3.1(
)zeo-transfected cells was greater compared with
treated, non-transfected and transfected controls. Activity in cells
treated with aldosterone and antisense was lower compared with that in
cells treated with aldosterone alone or in addition to sense
oligonucleotide. Similarly, DZA decreased SAHHase activity compared
with control.
 |
DISCUSSION |
The current study directly assesses the role SAHHase plays in the
signal transduction initiated by aldosterone which culminates in
increased Na+ reabsorption. Aldosterone increased the
activity of SAHHase through post-translational regulation. Moreover, as
shown by both pharmacological and molecular inhibitors, induction of
SAHHase activity was critical for aldosterone to increase and maintain
Na+ reabsorption across renal epithelial cells. The current
results also demonstrate that although overexpression of SAHHase
potentiates aldosterone-induced current and enzyme activity,
overexpression alone is insufficient to mimic completely all the
natriferic actions of the steroid. Thus, these results support the
hypothesis that the critical methylation required for aldosterone
signal transduction is regulated by SAH catabolism with SAHHase
activity being the control point.
Regulation of Na+ Transport by
Transmethylation--
Shown in Fig. 9 is
a schematic depiction of the critical enzymes and metabolites for the
aldosterone-induced transmethylation reaction that modulates
Na+ channel activity. Sariban-Sohraby and colleagues (4)
and Wiesmann et al. (5) were the first to document the
association of aldosterone-induced Na+ transport with the
methylation reaction. These investigators showed that methyl donors,
such as AdoMet, could mimic the actions of aldosterone. In addition,
inhibition of transmethylation blocked induction of transport by
aldosterone demonstrating that a critical methylation was necessary for
Na+ reabsorption. Interestingly, aldosterone increased
transmethylation of disparate classes of molecules, including both
proteins and lipids, suggesting that the mineralocorticoid regulated
methylation in general but did not regulate a specific transmethylation
reaction. The current results show that one mechanism through which
aldosterone regulates methylation is by altering the activity of
SAHHase. Our results demonstrate that aldosterone increases
Na+ transport by first increasing the activity of SAHHase
through an as yet undefined post-translational modification. An
aldosterone-induced increase in hydrolysis of SAH increases the rate of
most transmethylation reactions by reducing end product feedback
inhibition.

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Fig. 9.
Diagram showing the enzymes and metabolites
involved in methylation. Aldosterone activates SAHHase to decrease
cellular SAH. This relief of MTase from end product inhibition favors
the methylation of an unknown protein substrate (Pr). This
critical substrate methylation subsequently activates ENaC through a
mechanism not yet described. Also remaining to be described is the
specific, aldosterone-sensitive post-translational modification
regulating SAHHase activity.
|
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Several other investigators also have confirmed that Na+
transport and ENaC activity are regulated by transmethylation (3, 6-8); however, the substrate for this aldosterone-induced methylation remains unclear. Recent results from our laboratory suggest that methylation of p21ras is increased by
aldosterone (9), and results from the laboratory of Johnson (10)
support the notion that
ENaC also is an aldosterone-sensitive methylation substrate.
Characterization and Regulation of A6 Cell
SAHHase--
Deazaadenosine decreased aldosterone-induced SAH
hydrolysis (Fig. 3B) and SAHHase activity (Fig.
4A) in intact and cell-free assays, respectively. This
finding is consistent with that reported by others (11, 17). Similar to
DZA, antisense oligonucleotide complementary to the region around the
translation start site of xSAHHase mRNA decreased enzyme activity
(Fig. 5A).
The activity of SAHHase as measured by SAH hydrolysis in
steroid-deprived A6 cell lysate (Fig. 4A) was similar to
hydrolytic activity reported in crude extract of guinea pig cardiac
muscle (18) and bovine kidney (19); however, activity was greater compared with crude extract of cat, rabbit, rat, and dog heart (18).
Aldosterone repletion for 4 h to A6 cell monolayers doubled SAHHase activity to a level similar to that observed in rat liver extract (20). This induction of activity demonstrates that SAHHase is
regulated by aldosterone signal transduction, an observation consistent
with that reported previously by Finkelstein and Harris (21) showing
that adrenal steroids regulate SAHHase activity in rat epithelial
tissue. The mechanism for modulation of SAHHase by steroid, however,
remains to be determined.
Three mechanisms could account for increased activity in response to
aldosterone as follows: 1) increased enzyme concentration, 2) increased
activity through post-translation modification of the enzyme, and 3)
relief of the enzyme from metabolic regulation by increased degradation
of an inhibitor, such as adenosine. Aldosterone failed to significantly
increase SAHHase mRNA to the extent that SAHHase enzyme activity
increased (Fig. 4) suggesting that an increase in enzyme concentration
was unlikely. The observation that activity was higher in crude extract
from cells treated with aldosterone compared with untreated cells
suggests some form of post-translational modification, since SAHHase
presumably has been relieved of end product inhibition by the inclusion
of exogenous adenosine deaminase.
Translation of pxSAHHase3.1(
)zeo produced a protein of size (Fig. 1)
consistent with that reported previously for SAHHase (11, 19).
Overexpression of SAHHase increased enzyme activity in both HEK293
(Fig. 6A) and A6 cells (Fig. 7C). The observation that overexpressing A6 cells deprived of aldosterone had 2.65-fold increase in SAHHase mRNA levels compared with control transfectants but only 1.56-fold the activity supports the notion that
post-translational events regulated by aldosterone control enzyme
activity. Moreover, aldosterone application to overexpressing cells had
an additive effect on SAHHase activity demonstrating that distinct from
regulating protein number, steroid increased activity through some
other mechanism.
These results combined show that aldosterone increases SAHHase activity
through a signal transduction event culminating in post-translational
regulation of the enzyme. The primary amino acid sequence of SAHHase
contains multiple consensus phosphorylation sites for both
serine/threonine and tyrosine kinases. In addition, a number of
consensus sequences for fatty acylation exist. Although the mechanism
of SAHHase regulation by aldosterone remains to be determined, it is
possible that this enzyme is regulated by post-translational
modifications, such as phosphorylation or acylation. It will be
interesting to test whether SAHHase is regulated by the recently
characterized, aldosterone-induced serine/threonine protein
kinase, Sgk, which recently has been shown to be transcriptionally regulated in responsse to aldosterone (22).
Regulation of the Transmethylation Reaction by SAHHase
Activity--
It is well established that SAH is an end product
inhibitor of most transmethylation reactions involving AdoMet as the
methyl donor (11). Sariban-Sohraby and colleagues (4) first showed that
DZA and SAH inhibited the acute actions of aldosterone as measured by
Na+ flux across apical membrane vesicles prepared from A6
cells. Moreover, application of aldosterone increased both lipid and protein transmethylation in a DZA-sensitive manner in epithelia suggesting that aldosterone affected a general control point regulating all forms of methylation. Eaton et al. (3) have shown
through single channel analysis that DZA regulates Na+
channel activity likely through regulating the transmethylation reaction. Although all of these studies support the hypothesis that
SAHHase activity regulates substrate methyl esterification, this
mechanism was not the focus of any of these studies or investigated directly, and only a single pharmacological inhibitor was used. Moreover, DZA was used only at a single concentration in all previous studies. Because of this, nonspecific effects of DZA could not be ruled
out, and it was unclear if control of SAHHase was a physiological mechanism for regulating transmethylation reactions relevant to aldosterone-induced Na+ transport in epithelial cells.
The current study is the first to investigate directly the role SAHHase
plays in regulation of aldosterone-induced methylation and
Na+ current. The results of the current study show that
aldosterone addition to A6 cells increases both SAH and AdoMet
metabolism (Fig. 3B). Inhibition of SAHHase by DZA decreased
SAH metabolism to such an extent that cellular concentrations rose in
response to aldosterone treatment presumably because AdoMet metabolized by methyltransferase increased SAH, which could not be catabolized by
an inhibited SAHHase. This notion also can account for the decrease in
AdoMet metabolism observed in the presence of DZA with increased SAH
decreasing methyltransferase activity and, thus, AdoMet catabolism.
Methyltransferase activity in A6 cell lysate also was decreased by end
product inhibition demonstrating that SAHHase activity through
regulation of cellular SAH levels can control substrate methylation.
Overexpression of SAHHase resulted in an increase in both SAHHase
activity and substrate methyl esterification (Fig. 7C). These results are consistent with aldosterone increasing metabolism of
the end product inhibitor of methyl esterification reactions, SAH,
resulting in a concomitant increase in AdoMet metabolism in A6 cells.
Thus, in Na+-transporting epithelial cells, one regulatory
site controlling substrate methylation is modulation of SAHHase activity.
Regulation of Na+ Current by SAHHase
Activity--
Simultaneous addition of DZA with aldosterone for 4 h inhibited induced current in a dose-dependent manner
(Fig. 3A). Also, addition of DZA to monolayers reabsorbing
Na+ inhibited transport within 2 h in a
dose-dependent manner (Fig. 2). After 4 h,
Na+ current in the group treated with 30 µM
DZA began to return with current levels overshooting base line by
8 h. The time courses for maximal inhibition of existing and
aldosterone-induced transport are consistent with previous findings
also showing that DZA inhibits Na+ transport in toad
bladder and A6 epithelia (4, 5). Also consistent with our observations
that DZA inhibits aldosterone-induced Na+ reabsorption are
the findings of Eaton et al. (3) that application of DZA for
2-4 h inhibits aldosterone-induced activation of ENaC in cell-attached patches.
The results of the current study demonstrating that DZA blockade of
steady state Na+ reabsorption begins to recover after
4 h can be explained by metabolism of DZA to a less potent
inhibitor of SAHHase. It is likely that DZA, which also is a substrate
for SAHHase in the synthetic direction (11), was metabolized to
deaza-D-adenosylhomocysteine after 4 h. Since the
Ki for deaza-D-adenosylhomocysteine is
100 times that of DZA (23), this metabolism would enable current
recovery. Since DZA is absent in antisense experiments, results from
those experiments showing no current recovery are consistent with this mechanism.
Direct support for the hypothesis that aldosterone induces
Na+ transport, in part, by regulating SAH hydrolysis is
provided by antisense and overexpression experiments. Antisense
oligonucleotide but not sense decreased both aldosterone-induced enzyme
activity and Na+ current (Fig. 5) demonstrating that active
SAHHase is necessary for salt reabsorption. Overexpression of SAHHase
increased enzyme activity in an aldosterone-sensitive manner (Fig. 7).
Moreover, overexpression of SAHHase potentiated aldosterone-induced
current but did not affect basal currents (Fig. 8) suggesting that
increased SAHHase activity alone was insufficient to signal increased transport.
The current results support the hypothesis that a critical methyl
esterification in response to aldosterone application signals the
increase in ENaC activity at the apical membrane of epithelial cells.
Since methyltransferase and SAHHase are metabolically linked by SAH
concentration, the latter enzyme is a likely site for regulating methyl
esterification. The results of the current study demonstrate that
aldosterone first must increase SAHHase activity to initiate the
subsequent increase in both methyl esterification and Na+
transport. Thus, regulation of SAHHase activity by aldosterone-induced signal transduction is a control site for steroid induction of methylation and subsequent Na+ reabsorption.