Laboratory of Molecular and Cellular Physiology, School of Medicine, University Los Andes, Casilla 20106, Santiago 20-Chile
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ABSTRACT |
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Na+-K+-ATPase
gene expression and activity were studied in aortas from
adrenalectomized (ADX) rats and ADX rats with deoxycorticosterone supplement (ADX-DOCA). Northern analysis of RNA from ADX rats revealed
a significant decrease in
2-mRNA levels (38.5 ± 8.3% of control, P < 0.01) that was
prevented by DOCA (P < 0.05). A decrease to 55.8 ± 7.7% in
2-isoform protein was observed
8 days after adrenal removal (P < 0.05); DOCA reversed this effect (90.8 ± 10.5%). Adrenalectomy
induced a decrease of 68.5 ± 4.5% in
1-mRNA (P < 0.01) and 52.7 ± 8.3% in
ADX-DOCA rats (P < 0.01). Also, a
reduction in
1-isoform protein
that was not prevented by DOCA was detected after adrenalectomy (47.1 ± 11%, P < 0.01).
In contrast, no differences in
1-mRNA or -protein levels were
observed. Vascular sodium pump activity was reduced to 59.8 ± 4.6% of control values after adrenalectomy
(P < 0.01); this reduction was
reversed by DOCA. Our data indicate that corticosteroids regulate
Na+-K+-ATPase
isoform expression and activity in vascular tissue in vivo, suggesting
a mineralocorticoid-dependent modulation of
2-Na+-K+-ATPase
gene expression in aorta, with
1-isoform expression dependent on the presence of glucocorticoids.
vascular smooth muscle cells; aldosterone; hypertension
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INTRODUCTION |
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SODIUM-POTASSIUM-ADENOSINETRIPHOSPHATASE
(Na+-K+-ATPase)
of vascular smooth muscle cell (VSMC) membrane maintains the
electrochemical Na+-K+
gradient across the plasma membrane. The
Na+-K+-ATPase
is a heterodimeric protein, composed by a large -subunit of
100
kDa and a smaller glycosylated
-subunit of
55 kDa. All functional
sites involved in catalysis have been delineated to the
-subunit.
Four distinct
-isoforms have been identified in rat tissues, namely
1,
2,
3, and
4 (44, 45). The
-isoforms are encoded by separate genes, and their expression has been
demonstrated to be tissue specific and developmentally regulated (7, 9, 12, 21, 41). The
-subunit appears to be required for the
-catalytic function, and it is involved in the membrane integration of the
-subunits (1, 16). Three
-subunit isoforms
(
1,
2, and
3) have been cloned in rats
(17, 31, 32).
The critical contribution of the
Na+-K+-ATPase
activity to the modulation of intracellular
Na+ and
Ca2+ (10, 11, 13), vascular smooth
muscle tone (3), and peripheral resistance (37), and its role in the
pathogenesis of hypertension have been proposed by several
investigators (6, 8, 20, 22). Corticosteroid hormones have been
implicated in the regulation of the
Na+-K+-ATPase
in mammalian kidney and colon (53, 54), and the hypertensive effect of
high levels of corticosteroids with increments in total peripheral
resistance is well established. The presence of
high-affinity corticosteroid receptors in rat aorta (26, 39) and the
assessment of sodium pump activity in rat arteries under corticosteroid
treatment suggest molecular regulatory effects of adrenal
corticosteroid hormones on
Na+-K+-ATPase
activity of rat arteries. For example, dexamethasone has been shown to
increase Na+ pump activity in rat
tail arteries (19, 50). In the DOCA salt-hypertensive rat, increased
(46), decreased (42), and variable changes (47) in vascular
Na+-K+-ATPase
activity have been described. In addition, aldosterone infusion in rats
induced an increase in blood pressure, accompanied by an increase in
ouabain-sensitive sodium efflux in tail arteries (15). A recent study
of -catalytic subunit mRNA abundance in the aorta of DOCA
salt-hypertensive rats has shown a marked increase in
1- and
2-mRNA (48). Finally, in VSMCs,
both dexamethasone and aldosterone (38, 40) induced the
1- and
1-gene expression. However, the
hormonal effects on the expression of the
2-isoform were not evaluated in
this model because, contrary to the rat vascular tissue in vivo, the
cultured rat VSMCs exclusively express the
1-catalytic isoform (40).
We hypothesized that in vivo endogenous corticoids exert a regulatory
effect on sodium pump isoform expression affecting catalytic activity
in vascular smooth muscle. Previously reported work has focused on DOCA
salt-hypertensive animals, and, surprisingly, no report about the
effect of adrenalectomy on sodium pump expression in vascular tissue
has been published. Therefore, the purpose of the present work was to
identify the - and
-subunit isoforms present in the rat aorta and
to study in vivo the molecular regulatory effect of adrenal steroids on
the isoforms and activity of the Na+-K+-ATPase.
We used adrenalectomized animals (ADX) and ADX animals plus the
mineralocorticoid DOCA (ADX-DOCA). Our characterization studies by
RT-PCR and Northern blot demonstrated
1-,
2-, and
1-isoform expression in rat
aorta. The functional studies
[86Rb/K uptake in aortic
rings and K+-dependent
p-nitrophenolphosphatase membrane
activity (K+-pNNPase)]
demonstrated a diminished
Na+-K+-ATPase
activity in aortas from ADX animals. A recovery of the sodium pump
activity was obtained in ADX animals with 8-day DOCA-replacement therapy. These effects were associated with changes in gene expression and protein concentration of
2-
and
1-isoforms, as shown by Northern and Western blot analyses.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (130-150 g) were divided into three groups: ADX, ADX-DOCA, and a control group. The ADX animals were laparotomized under anesthesia with ether, and the adrenal glands were removed. The ADX-DOCA group was also adrenalectomized, and immediately after surgery, the animals were subjected to daily intramuscular injections of DOCA (0.5 mg/100 g body wt) during 8 days. All animals were fed with standard rat chow, and the ADX group received 0.9% NaCl (wt/vol). Control and ADX-DOCA groups were allowed to have tap water.
RNA isolation. The thoracic aorta was immediately processed after all adventitia were removed, and it was washed with sterile ice-cold 0.9% NaCl solution. The tissue of one to three animals was used for total cellular RNA extraction with the guanidinium thiocyanate or Trizol (GIBCO, Life Sciences) method as per specifications of the manufacturer. RNA concentration was determined by spectrophotometry in triplicate and used in RT-PCR or Northern blot studies.
RT-PCR studies. The initial
identification of - and
-subunit isoforms expressed in aorta of
control, ADX, and ADX-DOCA rats was performed by RT-PCR from total
cellular RNA. Rat skeletal muscle, kidney, or brain total cellular RNA
was used as a positive control. Aliquots of 5-10 µg of RNA were
incubated for 5 min at 65°C, transferred to ice, and centrifuged at
4°C for 1 min at 12,000 g
(4°C). For RT, 2- to 9-µl aliquots (0.3-2 µg) were
incubated in 5 mM MgCl2, 10 mM
Tris · HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 1 mM deoxynucleoside triphosphate, 1 U/µl AMV reverse transcriptase (Promega), 1 U/µl RNasin, and oligo(dT) primer or random hexamers at
42°C for 45 min following instructions by the manufacturer (Promega). The synthesized cDNA was immediately used or
stored at
20°C.
The cDNA isoform-specific fragments were amplified by PCR with rat
sequence-specific primers (20-25 nucleotides/primer). The fragments that were amplified were as follows:
1-fragment, corresponding to
nucleotides 418 to 1,437 of the
1-cDNA sequence obtained from Gene Bank (45);
2-fragment,
corresponding to nucleotides 450-1,762 of the
2-cDNA sequence (45);
3-fragment, corresponding to nucleotides 230-1,240 of the
3-cDNA sequence (45) and a
second
3-fragment corresponding
to nucleotides 18-335 of
3-cDNA sequence as described by
Lücking et al. (30);
4-fragment, corresponding to
nucleotides 1,036-1,443 of
4-cDNA sequence obtained from
Gene Bank (44);
1-fragment,
corresponding to nucleotides 443-1,464 of
1-cDNA sequence (56); and
2-fragment, corresponding to nucleotides 2,406-3,118 of
2-cDNA gene sequence obtained
from Gene Bank (24). PCR was performed with 25-40
cycles of the following temperatures: 95°C, 45 s; 60°C, 30 s;
and 72°C, 45 s (
1-,
2-, and
4-probes). The annealing
temperature was changed to 59°C for
3- and
2-probes and to 56°C for
1-probes. Optimum
MgCl2 concentrations were studied
for each primer set. PCR products were analyzed by electrophoresis on
1% ethidium bromide-stained agarose gels. In each case, a single band
of the predicted size was obtained.
Northern blot analysis. Equivalent
amounts (15-25 µg) of total cellular RNA were electrophoresed on
2.2 M formaldehyde-agarose gels, washed with 50 mM NaOH, blotted, and
fixed onto nylon membranes (GeneScreen Plus, Du Pont), as previously
described by Bonilla et al. (9). The membranes were hybridized to
1-,
2-,
3-, and
1-subunits of
Na+-K+-ATPase
cDNA probes. The cDNA probes were amplified by PCR with rat
sequence-specific primers (20-25 nucleotides/primer) as indicated in RT-PCR studies. Rat
aorta, skeletal muscle, or brain cDNA was used as a template for the
reactions. PCR products were analyzed by electrophoresis on 1%
ethidium bromide-stained agarose gels, purified with Wizard PCR
preparation (Promega), and labeled with a random primer labeling kit
(Promega). Hybridization was performed at 65°C for 24 h with 25 ng
2'-deoxycytidine
5'-[32P]triphosphate-labeled
probes (109 to 2 ×108 counts/min). Before
autoradiography, membranes were washed stringently two times for 5 min
with a solution of 2× saline-sodium citrate (SSC) plus 0.1% SDS
(1× SSC is 0.15 M NaCl and 0.15 M sodium citrate, pH = 7.0) and then two times for 15 minutes with a solution of 0.1×
SSC plus 0.1% SDS at 65°C. Films (Fuji RX) were placed in contact
with the membranes in cassettes containing intensifying screens and
were left exposed at
45°C for 24-72 h, and three to
four plates were used to ensure that we did not saturate the film. The
amount of mRNA present in each lane was quantified by computer scanning
densitometry analysis, comparing the intensity of the experimental
sample with the control rat mRNA sample. Standard curves were run to
work in the linear range. The values obtained were corrected by the
total cellular RNA loaded in each lane, as quantified by computer
scanning densitometry of 28S and 18S ribosomal RNA subunits in ethidium
bromide-stained pictures of gels before transfer (21).
Membrane preparation and Western blot
analysis. To minimize the potential selective
enrichment of different pump isoforms during the purification
procedure, we prepared a crude membrane fraction. Cleaned and washed
thoracic aortas from three animals were homogenized by a motor-driven
Potter-Elvehjem Teflon homogenizer in ice-cold buffer containing 50 mM
Tris · HCl, 1 mM phenylmethylsulfonyl fluoride
(PMSF), 2 µM leupeptin, 2 µM pepstatin, and 50 mM
-mercaptoethanol, pH = 7.4. The homogenate was centrifuged at 3,000 g for 10 min (4°C), and the
supernatant was centrifuged at 100,000 g for 45 min (4°C). The membranes
were suspended in 300 µl of 10 mM Tris · HCl, 10 mM
EDTA, 0.5 mM PMSF, 10% glycerol (vol/vol), and 50 mM
-mercaptoethanol (pH = 7.4) and stored at
20°C.
SDS-polyacrylamide gels were prepared according to the method of
Laemmli (27). The blotting procedures were according to Towbin et al.
(52). After blotting, the polyvinylidene difluoride membranes were
blocked with 5% nonfat milk in Tris-buffered saline (20 mM
Tris · HCl, 137 mM NaCl) plus 0.1% Tween
20. After washing, separate membranes were incubated with
mouse monoclonal anti-1 kindly
provided to us by Dr. M. Caplan; mouse monoclonal
anti-
2-McB2 kindly provided to
us by Dr Kathleen Sweadner; rabbit polyclonal anti-rat
1 (Upstate Biotechnology); or
rabbit polyclonal anti-
3 (Upstate Biotechnology). Blots were developed by enhanced
chemiluminescence (ECL+, Amersham) with horseradish
perioxidase-linked secondary antibodies. Films (Hyperfilm
MP, Amersham) were placed in contact with the membranes in cassettes
containing intensifying screens, and four to five plates were used to
avoid film saturation. Standard curves were run to work in the linear
range. The signal intensity present in each lane was quantified by
computer scanning densitometry analysis, comparing the intensity of the
experimental sample with the control rat sample lane.
Quantification dots on Western and Northern blots. Samples were quantified with a ScanMaker II HR (MICROTEK) interfaced to an IBM Aptiva computer with a Gel-Perfect C Program (9).
Sodium pump activity:
86Rb/K uptake in aortic
rings. The
Na+-K+-ATPase
activity was measured by ouabain-sensitive
86Rb/K uptake in aortic rings
according to Bofill et al. (7). Briefly, after the rats were
decapitated, the thoracic aorta was quickly removed and washed with
ice-cold Krebs-Ringer buffer (KRB) containing (in mM) 4.2 KCl, 1.19 KH2PO4,
120 NaCl, 25 NaHCO3, 1.2 MgSO4, 1.3 CaCl2, and 5 D-glucose (pH = 7.4).
The aorta was dissected free of connective tissue, and special care was
taken to avoid damage to the endothelium and rings (4-6 mm). At
the beginning of the experiments, aortic rings were equilibrated for 1 h in 2 ml of KRB at 37°C in a water-saturated atmosphere containing 95% O2-5%
CO2 in a Dubnoff incubator.
Thereafter, the aortic rings were equilibrated for 15 min in KRB
(37°C) in the presence of ouabain
(101 or
10
3 M), when indicated.
Finally, the tissues were incubated in 2 ml of KRB containing
86Rb (0.1 mCi/ml) in the presence
or absence of ouabain for 15 min, as described previously (18).
Transferring the aortic rings into ice-cold KRB stopped the reaction;
the tissue was then quickly washed in cold buffer and gently blotted.
Radioactivity of the samples was determined in triplicate by Cerenkov
radiation in a liquid scintillation counter in the presence of 0.1%
Tween 20 (2 ml).
Total pump activity was calculated by the difference between zero and
103 M ouabain. Low
ouabain-affinity activity was the
86Rb/K uptake in the presence of
10
5 M activity minus that
in 10
3 M ouabain. The high
ouabain-sensitive activity was calculated as the difference in
86Rb/K uptake activity in the
absence and presence of 10
5
M ouabain (18).
Sodium pump activity-enzyme activity in crude membranes. K+-pNPPase activity was measured according to the method described by Bers (4), with slight modifications. Crude membrane preparations from aorta were treated with 0.05% Triton X-100 for 20 min at 37°C and then diluted eightfold with cold buffer (1 mM EDTA, 10 mM Tris · HCl, pH 7.4). The reaction mixture (0.7 ml) contained (in mM) 20 NaCl plus 1 ouabain or 20 KCl, 20 MgCl2, 1 EDTA, 50 Tris · HCl (pH 7.4), and 30 µg of membrane protein. After a 5-min incubation at 37°C for temperature equilibration, the reaction was started by the addition of 100 µl p-nitrophenolphosphate (final concentration 5 mM). The reaction was terminated 35 min later by adding 50 µl 50% TCA and cooling on ice. After the samples were centrifuged to remove precipitated protein, 100 µl of 10 N NaOH were added to the supernatant. Absorbance at 410 nm was recorded, and K+-pNPPase activity was calculated as the difference in values observed in the presence of K+ minus the values observed in the presence of Na+ and ouabain. A standard curve from 5 to 60 nM p-nitrophenolphosphate was routinely run.
Statistical analyses. Values are
expressed as means ± SE. Differences between mean values of
densitometry data, K+-pNPPase, and
86Rb/K uptake were assessed by
Student's t-test (unpaired) or by one-way ANOVA when appropriate. Differences were accepted as
significant at the P 0.05 level.
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RESULTS |
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Expression of catalytic subunit isoforms of
Na+-K+-ATPase.
To elucidate the -subunit isoforms that are present in the rat
aorta, we conducted Northern, Western, and PCR studies in isolated
tissue from control, ADX, and ADX-DOCA rats. As shown below, we
confirmed the presence of
1-
and
2-isoforms by Northern and
Western measurements.
As a first approach to assess
3-expression in rat aorta, we
performed Northern blot studies on aortic total RNA and brain total RNA
that serve as a positive control (Fig.
1A).
No hybridization signal was observed with the
3-specific cDNA probe in aortic RNA isolated from control, ADX, and ADX-DOCA animals. As expected, an
intense hybridization to a 3.8-kb band in brain RNA was observed, showing the specificity of the probe. Second, the presence of
3-mRNA in rat aorta was also
checked by the more sensitive RT-PCR method. Figure
1B shows the results obtained with two
different sets of specific
3-subunit primers. No mRNA
transcripts were detected in four experiments with variable amounts of
aortic RNA used in the RT assay (0.1-2.0 µg) or with 30-40
amplification cycles. Also, no product was detected when studying total
RNA obtained from aorta of ADX or ADX-DOCA animals. As a positive control, we used brain total RNA, obtaining a strong band of PCR product of the expected size after 30 cycles of amplification. A third
independent approach to detect
3-isoform expression was to
identify the protein in rat aorta. Western blot and immunocytochemical studies performed with specific monoclonal antibodies (Upstate Biotechnology) did not detect
3-subunit protein in crude
aortic membranes (100 µg) or in aortic sections from control animals (data not shown). All of these results indicate no
3-isoform expression in rat
aorta, or at least
3-expression
at very low levels, under the detection limits of our assays. As far as
we know, no
3-isoform
expression has been detected in rat vascular endothelial cells (57).
Only one paper (43) was found that showed the presence of
3-isoform in rat tail artery.
Considering that the tail artery is a thermoregulator organ in rat,
this could reflect a functional difference in regional
-catalytic
expression of rodent vascular tissue. However, our control experiments
also failed to detect
3-expression in rat tail artery
at the mRNA level, suggesting that this isoform is not significantly
expressed in rat arteries.
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To assess -expression, RT-PCR studies were carried
out. No
4-mRNA was detected in
four different experiments with 0.3-2.0 µg of total RNA from
aorta of control, ADX, and ADX-DOCA animals. Also, no
4-product was obtained with 30 to 40 amplification cycles (Fig.
1C). As a positive control, we used
testis total cellular RNA (0.3 µg), obtaining a strong band of
product of the predicted size. The RT reaction efficiency was
determined by amplification of glyceraldehyde-3-phosphate-dehydrogenase
cDNA, demonstrating that the absence of
4-product in rat aorta did not
result from RT failure.
Effect of adrenalectomy on catalytic
Na+-K+-ATPase
isoform gene expression. We studied
1- and
2-isoforms from thoracic aorta of control, ADX, and ADX-DOCA rats. Each specific cDNA probe was hybridized to separate Northern blots containing equivalent amounts of
total RNA in each lane. The relative amount of each isoform was
determined and normalized to the control value.
The effect of adrenalectomy in rat aortic gene expression of
1-Na+-K+-ATPase
isoform at 8 days is shown in Fig. 2. Also,
the effect of DOCA-replacement therapy was studied. As expected, the
1-cDNA probe hybridized with a
3.7-kb mRNA transcript. The densitometric analysis of each blot
revealed that no significant changes in
1-mRNA relative amounts were
found in tissues from ADX animals or in ADX-DOCA rats when compared
with controls (Fig. 2B).
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Figure 3A
shows the effect of adrenalectomy in
2-catalytic isoform expression
at 8 days. As previously described, the probe recognized two
transcripts of 5.3 and 3.4 kb corresponding to mRNAs encoding the
2-subunit (7, 41). The
densitometric analysis demonstrated that the adrenalectomy induced a
decrement in
2-isoform gene
expression levels (Fig. 3B). At 8 days, the
2-mRNA density was
38.5 ± 8.3% of control level (P < 0.001). The DOCA administration to the ADX animals almost prevented
the reduction in
2-mRNA levels
(84.7 ± 5.6% of control value). A significant difference was
observed between ADX rats without or with DOCA,
P < 0.001, ADX vs. ADX-DOCA.
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Effect of adrenalectomy on
1-isoform gene
expression. A dramatic effect of adrenalectomy was
observed in
1-gene expression in the vascular tissue. As demonstrated by Northern blot studies in the
ADX group, these transcripts diminished to 31.5 ± 4.5% of the
control level at 8 days (P < 0.001, control vs. ADX). The group that received DOCA replacement had 47.3 ± 8.3% of the basal
1-mRNA
levels [P < 0.001, control vs.
ADX-DOCA; nonsignificant (NS), ADX vs. ADX-DOCA; see Fig.
4, A and
B].
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Finally, the expression of
2-subunits was also analyzed in
total RNA extracted from aorta of control, ADX, and ADX-DOCA animals by
RT-PCR. For
2-expression
studies, rat brain RNA and soleus total RNA were used as positive and
negative controls, respectively (51). A strong product band of the
expected size was obtained when 0.6 µg of brain RNA were used (Fig.
4C). No product was observed in
reactions performed with aortic or soleus total RNA (8 different experiments) varying the RNA amount used in RT reaction (0.3-3.0 µg) or PCR number of cycles (30-40). Again, the RT efficiency
was confirmed by glyceraldehyde-3-phosphate-dehydrogenase-specific cDNA
amplification; see Fig. 4C.
Effect of adrenalectomy and DOCA replacement in
isoform protein concentration. In the long-term
regulation of the
Na+-K+-ATPase,
the overall abundance of functional pumps is controlled not only at the
levels of transcription and transcript stability but also at the
translational and posttranslational levels. Therefore, we decided to
measure and compare the relative levels of the
1-,
2-, and
1-isoforms present in rat
aortic membranes by Western blot.
As expected, a band that migrates with an apparent molecular weight of
100,000 was recognized by the
anti-1-specific monoclonal antibody (Fig.
5A). No
differences in
1-protein
subunit amounts were found at 8 days among groups (Fig
5B). However, as shown in Fig.
6, the
2-catalytic subunit declined to
55.8 ± 7.7% of the control levels 8 days after adrenalectomy
(P < 0.01). The DOCA-replacement
therapy in ADX animals prevented the reduction in
2-protein amount (90.8 ± 10.5% of control level, P < 0.01, ADX vs. ADX-DOCA).
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The 1-subunit protein levels
were assessed in crude aortic membranes at 8 days. The results are
shown in Fig. 7. Compared with control
membranes, a marked decrease in ADX group was observed (41.6 ± 10%, P < 0.001). The administration
of DOCA to ADX rats was not able to restore protein levels that were
similar to ADX group (47.1 ± 11%);
P < 0.001, control vs. ADX-DOCA; NS,
ADX vs. ADX-DOCA.
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Regulatory effect of adrenal steroids in Na+-K+-ATPase activity. The regulatory effect of adrenal steroids and DOCA on the sodium pump activity of rat aorta was evaluated by two different approaches: K+-pNPPase in isolated membranes and ouabain-sensitive 86Rb/K uptake on intact aortic rings.
The K+-pNPPase activity in aortic
membranes of control rats was 2.8 ± 0.22 nmol · mg
protein1 · min
1.
The ADX group showed a significant reduction to 57 ± 14% (1.6 ± 0.39 nmol · mg
protein
1 · min
1,
P < 0.05, control vs. ADX). The
DOCA-replacement therapy prevented this decrement (2.6 ± 0.22 nmol · mg
protein
1 · min
1;
NS, control vs. ADX-DOCA; P < 0.05, ADX vs. ADX-DOCA).
To evaluate the Na+-K+-ATPase activity on intact aortic rings, we measured the ouabain-sensitive 86Rb/K uptake. As shown in Table 1, a significant decrease in the ouabain-sensitive 86Rb/K uptake was observed in ADX rats (59.8 ± 4.6% of control rats, P < 0.01, control vs. ADX); this uptake reflects the total Na+-K+-ATPase activity on intact tissue. DOCA replacement reversed the diminished Na+-K+-ATPase-mediated uptake observed in aortic rings from ADX rats (92.6 ± 9.1% of the control group), confirming the results obtained when measuring K+-pNPPase activity in membranes (P < 0.01, ADX vs. ADX-DOCA; NS, control vs. ADX-DOCA).
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In rats, the -isoforms differ in ouabain sensitivity:
1 is ouabain resistant, whereas
2 and
3 are highly sensitive to the
drug; consequently, the
-catalytic isoforms of vascular tissue could
be functionally distinguished with high
(10
3 M) or low
(10
5 M) ouabain
concentrations (31). The results of such experiments are included in
Table 1. As shown, in control tissue, the
86Rb/K uptake mediated by the
ouabain-sensitive component was ~30% of the total pump activity,
whereas in aortic rings from ADX rats there is a disappearance of the
high ouabain-affinity component (P < 0.001, ADX vs. control). Interestingly, the disappearance of this
activity after adrenalectomy was prevented by DOCA treatment. In fact,
in the ADX-DOCA groups, the ouabain-sensitive activity was similar to
control rats. On the other hand, no differences in the low
ouabain-affinity-mediated 86Rb/K
uptake were found between groups. These results in the
86Rb/K uptake mediated by the high
and low ouabain-affinity components are in agreement with the pattern
of expression of
1- and
2-catalytic isoforms of the
Na+-K+-ATPase.
DOCA treatment to control rats produced no statistically significant
change in total
Na+-K+-ATPase
activity (control rats, 167.1 ± 15.2 nmol
86Rb/K · g wet
wt1 · min
1;
control + DOCA, 183.2 ± 9.6 nmol
86Rb/K · g wet
wt
1 · min
1;
n = 8 rats).
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DISCUSSION |
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The results of the present study indicate that endogenous adrenal
hormones participate in the molecular regulation of
Na+-K+-ATPase
activity in vascular tissue. We found that
1-,
2-, and
1-subunit isoforms are
expressed in rat aorta and that the adrenalectomy reduced the sodium
pump activity, differentially affecting sodium pump isoform gene
expression. ADX rats were provided with drinking water containing 0.9%
NaCl to achieve volume balance and the survival of the ADX animals.
Soszynski et al. (48) have shown that varying the sodium intake from a
low to a high salt intake produced no statistically significant change
in the mRNA abundance for any of isoforms in different tissues,
including rat aorta.
Protein and RNA analyses did not detect
3- and
4-isoform expression in aorta,
suggesting, in principle, the existence of at least two different
Na+-K+-ATPase
isozymes in rat aorta,
1/
1
and
2/
1.
The existence of these isozymes in vivo is supported by the fact that
in several experimental systems it has been possible to form active
combinations of
1- and
2-isoforms with
1-subunits (5, 12).
Furthermore, it has been shown that nanomolar concentrations of ouabain
augment caffeine-evoked contractions in rat arteries (55), and previous studies have shown the existence of low- and high-affinity inhibition components of ouabain-sensitive
86Rb/K-uptake in rat aorta (31).
Altogether, the present experimental evidence is consistent with the
functional expression of
1- and
2-isoforms in vascular tissue.
The physiological role for multiple sodium pump isozymes in different
tissues is actually unknown. It is possible that in rat aorta in vivo
the different isozymes could be targeted to defined cellular domains,
allowing the cell to respond to different demands. In fact, Juhaszova
and Blaustein (23) evidenced that the
1-isoform was ubiquitously
distributed all over the plasma membrane in astrocytes, neurons, and
arterial myocytes in culture, and the ouabain-sensitive isoforms
(
2 and
3) were confined to a
reticular distribution within the plasma membrane that paralleled underlying endoplasmic or sarcoplasmic reticulum. The above authors suggest that
1 could regulate
bulk cytosolic Na+ concentration,
whereas
2 and
3 might regulate
Na+ and indirectly
Ca2+ in a restricted cytosolic
space between the plasma membrane and the reticulum. It is well
established that rat
1-subunit
shows a slightly lower Michaelis constant
(Km) for
Na+ and lower affinity for cardiac
glycosides than
2, making this hypothesis very attractive. However, the characterization of isozyme compartmentalization in vascular tissue in vivo remains to be determined.
It is well established that adrenal steroids control the synthesis of
Na+-K+-ATPase
in renal tubules and colon-stimulating
1-gene expression (14, 54).
Previous studies on the effect of corticosteroids in VSMCs showed a
significant increment in
1-expression by aldosterone (40) and dexamethasone (38). In contrast, our present study shows that
adrenalectomy did not induce changes of
1-Na+-K+-ATPase
expression in rat aorta. Moreover, DOCA-replacement therapy did not
affect
1-mRNA or -protein
levels in aorta. These results indicate that factors other than adrenal
hormones also participate in
1-pump regulation in vivo,
eliciting the upregulation of
1-catalytic subunit in ADX rats.
Different from 1-gene
expression, we found a dramatic downregulation of
2-mRNA and -protein levels in
ADX rats. This was prevented by DOCA-replacement therapy, suggesting a
stimulatory role of mineralocorticoids in
2-gene expression. The effect
of DOCA was maximal at 8 days, but it was also present after 4 days of
treatment in the ADX rats (data not shown).
The 86Rb/K uptake studies showed a
marked decrease of
Na+-K+-ATPase
activity in aorta from ADX animals, and this activity was recovered by
DOCA treatment. A similar result was obtained when measuring
Na+ pump activity in crude
membranes from ADX and ADX-DOCA animals. These results are consistent
with the observation presented above on mRNA and protein levels and
consistent with earlier observations showing that corticosteroids
produce an upregulation of the enzyme activity in cardiac tissue (25)
and that adrenalectomy decreased the total
Na+-K+-ATPase
activity (49). Altogether, our data suggest that
2-gene expression in aorta is
adrenal (corticosteroid)-dependent and that the sustained presence of
endogenous corticoids is required for normal aortic
2-expression.
On the other hand, adrenalectomy induced a marked downregulation of
1-mRNA and -protein, suggesting
that endogenous levels of adrenocortical hormones are required for
normal
1-subunit expression.
Previous reports indicate a substantial increment in
1-mRNA levels in VSMC cultures
on the addition of corticosteroids (38, 40). However, no significant
changes in
1-mRNA and protein amounts were observed in the ADX-DOCA rats compared with ADX animals. These results indicate that the in vivo modulation of
1-mRNA and protein levels in
aorta could be mediated by glucocorticoids or coordinated stimulation
of gluco- and mineralocorticoids, because DOCA alone was unable to
restore either
1-mRNA or
protein abundance.
Interestingly, the recovery of
Na+-K+-ATPase
activity observed in ADX-DOCA animals was correlated with
2-mRNA and -protein restoration, but no significant effect over
1-subunit expression was found.
Assuming that the assembly of
/
-heterodimer is a prerequisite for
Na+-K+-ATPase
function, these results suggest that under normal conditions in rat
aorta the pool of
1-subunits is
larger than the
-isoforms pool. Thus only a profound diminution in
1 could affect
1/
1- and
2/
1-isozyme
function. This hypothesis is supported by observations that in
mammalian renal cells in culture,
1 can mature in excess of
1-complements, forming an
unassociated pool of
-subunits (35, 36). Similarly, a
disproportionate abundance of
- compared with
1-mRNA subunit content in
canine vascular smooth muscle has been observed (2).
Also, in rat skeletal muscle, an overall excess of
- over
-subunit proteins has been reported (28). Another
possibility is that DOCA treatment could have a significant effect in
the
3-subunit, which might
compensate for the lack of effect of DOCA treatment on
1-subunit.
In summary, we found 1-,
2-, and
1-isoform expression in rat
aorta. Adrenalectomy induced a marked decrease of
Na+-K+-ATPase
activity in vascular tissue, with a concomitant decrease in
2- and
1-gene expression. In contrast,
1-isoform expression was
unchanged. The DOCA-replacement therapy in ADX rats did not modify
1-levels but elicited an
upregulation of
2-catalytic isoform expression and recovery of the sodium pump activity, suggesting that mineralocorticoids increase the
Na+-K+-ATPase
activity through the upregulation of
2-pump expression. The DOCA
replacement did not produce a significant effect over the reduced
1-subunit abundance in ADX
rats, and the participation of glucocorticoids in the regulation of
1-isoform gene expression in
vascular tissue needs to be characterized further.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. Caplan (Yale Medical School) and Dr. Kathleen
Sweadner (Harvard Medical School) for kindly providing the
anti-1- and
anti-
2-monoclonal antibodies,
Dr. Valeska Vollrath (Pontificia Universidad Católica de Chile)
for advice in molecular biology studies, and Javier Venegas for
technical assistance. Dr. Nelson Ruiz-Opazo kindly reviewed the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by FONDECYT Grant 197-0696.
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.
Address for reprint requests: L. Michea, Laboratory of Molecular and Cellular Physiology, School of Medicine, Univ. Los Andes, Casilla 20106, Santiago 20-Chile.
Received 29 April 1998; accepted in final form 7 August 1998.
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