Departments of Internal Medicine and Integrative Biology, Pharmacology, and Physiology, University of Texas Medical School at Houston, Houston, Texas 77030
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ABSTRACT |
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Nitric oxide (NO)
has been implicated as an autocrine modulator of active sodium
transport. To determine whether tonic exposure to NO influences active
sodium transport in epithelial cells, we established transfected
medullary thick ascending limb of Henle (MTAL) cell lines that
overexpressed NO synthase-2 (NOS2) and analyzed the effects of
deficient or continuous NO production [with or without
NG-nitro-L-arginine methyl ester
(L-NAME) in the culture medium, respectively] on
Na+-K+-ATPase
function and expression. The NOS2-transfected cells
exhibited high-level NOS2 expression and NO generation, which did not
affect cell viability or cloning efficiency.
NOS2-transfected cells were grown in the presence of
vehicle,
NG-nitro-D-arginine
methyl ester (D-NAME), or
L-NAME for 16 h, after which
86Rb+
uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The
NOS2-transfected cells allowed to
produce NO continuously (vehicle or
D-NAME) exhibited lower rates of
ouabain-sensitive 86Rb+
uptake (~65%), lower levels of
Na+-K+-ATPase
1-subunit mRNA (~60%), and reduced rates of de novo
Na+-K+-ATPase
1-subunit transcription compared with
L-NAME-treated cells. These
results have uncovered a novel effect of NO to inhibit transcription of
the
Na+-K+-ATPase
1-subunit gene.
sodium pump; nitric oxide synthase; gene expression; kidney; sodium transport
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INTRODUCTION |
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NITRIC OXIDE (NO) is a free radical gas that exerts potent effects on essential biological functions (36), including the regulation of ion transport in epithelia (22, 46). Recent data in animals and humans have documented that NO plays a critical role in the homeostatic control of renal sodium excretion and the extracellular fluid volume (23). Intrarenal NO synthesis is increased during periods of increased salt intake to facilitate natriuresis and maintain normal blood pressure (31, 41, 44, 49). Conversely, impaired NO synthesis or action appears to contribute to the maladaptive renal sodium handling that results in salt-sensitive hypertension (16, 53). Although many of these responses appear to be mediated by direct or indirect actions on the renal microcirculation, NO also exerts direct effects on solute and water transport by the renal tubules. For example, in vivo animal studies have shown that acute or chronic administration of NO synthase (NOS) inhibitors to animals, at concentrations that do not measurably affect glomerular or systemic hemodynamics, impairs urinary sodium excretion (41, 44, 49), whereas NO donors promote natriuresis (30). In humans, chronic administration of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) resulted in a 40% reduction in fractional excretion of sodium (3).
In vitro studies with NOS inhibitors and NO donors have shown that NO
can inhibit the functional activity of transport proteins involved in
transepithelial Na+ reabsorption
(46). For example, NO inhibited
Na+ entry in the cortical
collecting duct (45) and
Na+/H+
exchange in the proximal tubule (40, 50). NO was also shown to inhibit
Na+-K+-ATPase
activity in renal medullary slices (32), kidney enzyme preparations
(6), and mouse proximal tubule cells that had been stimulated with
lipopolysaccharide (LPS) and interferon (IFN)- (14). The mechanisms
by which NO donors inhibit
Na+-K+-ATPase
activity in renal tissues include cGMP-dependent and -independent mechanisms (14, 32). The cGMP-independent effects on enzymatic activity
might be mediated by the blockade of sulfhydryl groups within the
enzyme, because the sulfhydryl reagents dithiothreitol and cysteine
restored
Na+-K+-ATPase
activity and thiol content after exposure to thiol-containing NO
derivatives (6). The possibility that NO might act at more proximal
steps of
Na+-K+-ATPase
gene expression, such as gene transcription, however, has not been
explored despite the fact that NO is known to influence the activities
of transcription factors (7, 27, 38, 43, 47) that commonly regulate
other genes.
To investigate the effects of constitutive NO production on the
Na+-K+-ATPase,
and by inference, transepithelial sodium transport, we used gene
transfer to overexpress the gene encoding murine NO synthase-2 (NOS2;
also known as "inducible" NOS) in a medullary thick ascending
limb of Henle (MTAL) cell line that lacks basal NOS expression (25).
This strategy eliminated the myriad of confounding cellular effects of
immunoactive agents needed to induce
NOS2 gene expression, allowed
endogenously produced NO to interact with cellular constituents, and
permitted the analysis of
Na+-K+-ATPase
biosynthesis and function at multiple levels. The results indicate that
endogenously produced NO inhibits the expression and activity of the
Na+-K+-ATPase,
in large part, by constraining
Na+-K+-ATPase
1-subunit gene transcription. This previously unrecognized effect of
NO may contribute to the known natriuretic and diuretic effects of NO
in vivo.
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MATERIALS AND METHODS |
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Reagents.
L-Glutamine, heat-inactivated
fetal bovine serum (FBS), penicillin, streptomycin, G418, and DMEM
lacking phenol red were from GIBCO (Grand Island, NY). LPS from
Escherichia coli O111:B4,
NG-nitro-D-arginine
methyl ester (D-NAME), and
L-NAME were from Sigma Chemical
(St. Louis, MO). Radiochemicals were purchased from Amersham (Arlington
Heights, IL). The Tfx-50 reagent was purchased from Boehringer Mannheim
(Germany). RNAzol II was acquired from Tel-Test (Friendswood, TX).
Mouse monoclonal antibody (MAb) against murine NOS2 was from
Transduction Laboratories (Lexington, KY). Mouse MAb against
Tamm-Horsfall glycoprotein was provided by Dr. John Hoyer (Children's
Hospital, Philadelphia, PA). Affinity-purified antipeptide IgG directed
against the 5-hydroxytryptamine
5-HT1A receptor was obtained from
Dr. John Raymond (Duke University, Durham, NC). Mouse recombinant
IFN- was from Genzyme (Cambridge, MA).
Cell culture.
The MTAL-derived cell line ST-1 (15, 25), a gift from Dr. Adam Sun, was
maintained in DMEM supplemented with 10% FBS, 50 U/ml penicillin, and
50 µg/ml streptomycin (complete medium). The mouse macrophage cell
line RAW 264.7 (American Type Culture Collection) was grown in complete
medium. For induction of NOS2, RAW 264.7 cells were treated with LPS
(100 ng/ml) and IFN- (0.5 U/ml) for 16 h before study.
Transfection procedures and selection of clones. The encoding DNA for murine NOS2 (25) was cloned into the Hind III and Sal I sites of the mammalian expression vector pcDNA3.1 (Invitrogen) downstream of the cytomegalovirus (CMV) promoter to yield the recombinant molecule pcDNA3.1/NOS2. Subconfluent ST-1 cells were grown on 10-mm culture dishes and transfected with pcDNA3.1 (as a vector control) or pcDNA3.1/NOS2 with the Tfx-50 reagent. Briefly, 10 µg of plasmid DNA and 22 µl of Tfx-50 reagent were mixed with 6 ml DMEM. The mixture was added to the monolayers and incubated for 2 h at 37°C in a 5% CO2 incubator. Twelve milliliters of prewarmed complete medium were then added to the cultures. After 48 h, the medium was replaced with complete medium containing 5 mM L-NAME and 600 µg/ml G418. L-NAME (5 mM) was included in the culture medium during the selection process to inhibit the activity of the overexpressed NOS2 enzyme, thus eliminating potential NO cytotoxicity during the selection of individual clones.1 The L-NAME-G418 medium was replaced every 3 days until individual resistant colonies were isolated and established in culture as individual lines. All lines were maintained in L-NAME-G418 medium and frozen after one to three in vitro passages. For the identification of NOS2 gene-positive cells, cells of each clonal cell line were plated into individual wells of 24-well plates. Once confluent, the cells were washed and incubated with complete medium lacking L-NAME. After specified times, culture supernatants were collected for nitrite analysis. We selected pcDNA3.1/NOS2-transfected cell lines that expressed functional NOS2, as determined by nitrite assays and immunoblot analysis, for further study.
Nitrite measurements. Nitrite, the stable metabolite of NO, was measured in culture supernatants by a modification of the Griess reaction (25).
Preparation of cell lysates and immunoblot
analysis.
Cell monolayers were washed twice with 10 ml ice-cold homogenization
buffer (250 mM sucrose, 50 mM Tris · HCl, pH 7.4, 1 mM EGTA) and then scraped into 3 ml homogenization buffer.
The cells were pelleted by centrifugation, and the pellet was
resuspended in 1 ml lysis buffer (140 mM NaCl, 10 mM
Tris · HCl, 1.5 mM
MgCl2, 0.5% NP40, pH 8.6) and
incubated on ice for 5 min. Homogenates were centrifuged at 10,000 g for 20 s at 4°C, and the
resulting supernatants were collected, stored, and frozen at
80°C until used. The protein concentrations of the lysates
were measured by the bicinchoninic acid (BCA) assay (Pierce, Rockford,
IL). Twenty-milligram samples were diluted in
electrophoresis sample buffer, boiled for 5 min, and electrophoresed
through 0.1% SDS-7.5% polyacrylamide gels. The proteins were
electrophoretically transferred to nitrocellulose membranes (Hybond
ECL; Amersham). Equality of sample loading and blotting was verified by
Ponceau S staining of the membranes. The blots were quenched in
blocking solution (1% BSA in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1%
Tween 20) for 1 h at room temperature, incubated for 1 h at room
temperature with antibodies (5 µg/ml) against NOS2, Tamm-Horsfall
glycoprotein, or the 5-HT1A
receptor in blocking solution, and then washed extensively with a
solution containing 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween
20. The antigen-antibody complexes were detected by the enhanced
chemiluminescence protocol (ECL; Amersham) using horseradish
peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG, as appropriate.
Indirect immunofluorescence microscopy.
ST-1 cells were plated on glass coverslips in 35-mm culture dishes and
grown to confluence. Cells were fixed and permeabilized with
methanol:acetone (1:1) at 20°C for 10 min, blocked in PBS containing 1% BSA (PBS-BSA) for 1 h at room temperature, and incubated for 1 h at room temperature with 0.5 µg/ml anti-NOS2 antibody in
PBS-BSA. The primary antibody was removed, and, after three washes in
PBS-BSA, the cells were incubated with 5 µg/ml FITC-conjugated goat
anti-mouse IgG in PBS-BSA at room temperature for 1 h. The cells were
washed for 30 min in several changes of PBS-BSA and mounted on glass
slides in mounting medium. The sections were examined and photographed
with a Nikon Labophot inverted fluorescence microscope.
86Rb+ uptake. Uptake of 86Rb+, a K+ congener, was measured in cells grown in 24-well plates according to a published protocol (24). Cells grown on 24-well plates in the presence of vehicle, D-NAME, or L-NAME for 16 h were preincubated at 37°C for 30 min with assay buffer containing (in mM) 140 NaCl, 1 KCl, 5.5 glucose, 1 MgSO4, 2 CaCl2, 2.5 NaH2PO4, and 10 HEPES, pH 7.4, containing vehicle, 1 mM ouabain, 0.1 mM Sch-28080, 0.2 mM bumetanide, or combinations of the inhibitors as indicated in RESULTS. Uptake was initiated by the addition of 0.2 ml uptake buffer containing ~1 µCi/ml 86Rb+ and vehicle or transport inhibitor (ouabain, Sch-28080, bumetanide) to each well. After 15 min at 37°C, the uptake was stopped by six rapid washes with ice-cold stop buffer (100 mM MgCl2, 10 mM Tris-HEPES, pH 7.4). Parametric studies indicated that this time point was in the linear range of uptake. The cells were solubilized in 2% SDS-0.1 N NaOH, and the resulting extracts were measured for 86Rb+ by Cherenkov radiation. To determine the absolute rates of bumetanide-sensitive and ouabain-sensitive 86Rb+ uptake, we assayed the extracts for protein content by the BCA protein assay reagent (Pierce), and normalized the uptake rates to these values. For the experiments to test the potential inhibitory effects of Sch-28080, the data were normalized to the wells. Triplicate or quadruplicate measurements were obtained in each uptake condition and represent a single observation.
Northern analysis.
ST-1 cells were grown on 150-mm plates in the presence of vehicle,
D-NAME, or
L-NAME for 16 h at 37°C.
Thereafter, RNA isolation and Northern blotting were performed
according to our previously published methods (24), except that the
blots were sequentially hybridized with
32P-labeled cDNA probes specific
for the rat
Na+-K+-ATPase
1-subunit (nucleotides 3207-3529) (8) and rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (nucleotides
469-984) (24) were used. The blots were stripped for analysis of
Na+-K+-ATPase
1-subunit mRNA before proceeding to the GAPDH analysis. The blots
were washed to a final stringency of 0.1× SSC, 0.1% SDS (where
1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 60°C.
Autoradiographs of the blots were prepared at 70°C.
Nuclear run-on transcription.
ST-1 cells were grown on 150-mm plates in the presence of vehicle,
D-NAME, or
L-NAME for 16 h at 37°C.
Isolation of nuclei and the nuclear run-on transcription assay in the
presence of [-32P]UTP were
performed as previously described (34). cDNA inserts specific for rat
Na+-K+-ATPase
1 (nucleotides 3207-3529) and rat
-actin (nucleotides 2498-2765) were denatured and immobilized to nitrocellulose
membranes with a dot-blot apparatus. Equal incorporated counts of
32P-labeled nuclear RNA were
hybridized to the membranes at 60°C for 24 h, after which the
filters were washed and exposed to X-ray film at
80°C for
72 h.
Data analysis. The intensities of bands on the autoradiograms were measured by whole-band densitometry software running on a SPARC Station IPC (Sun Microsystems, Mountain View, CA) equipped with an image analysis system (Bio Image, Ann Arbor, MI). Quantitative data are presented as means ± SE and were tested for significance by ANOVA. For P < 0.05, we used the post hoc Bonferroni-Dunn test to establish significance.
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RESULTS |
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Additional phenotypic characterization of ST-1
cells.
We previously reported that, like the MTAL in vivo, ST-1 cells express
immunoreactivity for the 5-HT1A
receptor and Tamm-Horsfall glycoprotein (25). To determine whether ST-1
cells also express a bumetanide-sensitive
K+ uptake mechanism typical of the
MTAL in vivo, we measured
86Rb+
uptake of confluent monolayers of ST-1 cells in the presence and
absence of 0.2 mM bumetanide. Bumetanide inhibited
86Rb+
uptake by ~40% compared with vehicle-treated controls (16.6 ± 2.0 vs. 27.6 ± 1.8 nmol · mg
protein1 · min
1,
respectively; n = 4, P < 0.05). This result is compatible
with and extends the scope of earlier work showing that ST-1 cells express [3H]bumetanide
binding sites at their apical membranes (15).
Transfection and selection of NOS2-expressing ST-1 cells. To establish stable transfectants, we transfected ST-1 cells with either pcDNA3.1 (as a vector control) or pcDNA3.1/NOS2 plasmids and selected them in medium containing G418. We switched G418-resistant clones to L-NAME-free medium and screened them for nitrite production and for NOS2 protein expression by Western blotting and indirect immunofluorescence microscopy using an anti-NOS2 MAb.
As shown in Fig. 1A, ST-1 cells transfected with pcDNA3.1 produced negligible amounts of nitrite, whereas the pcDNA3.1/NOS2-transfected cells tonically produced micromolar amounts of nitrite. The nitrite production by the pcDNA3.1/NOS2-transfected cells was virtually abolished by 5 mM L-NAME, indicating that the nitrite was produced from NOS.
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Effect of endogenous NO production on
Na+
pump activity.
To test the effects of tonic NO production on
Na+-K+-ATPase
function, we grew
pcDNA3.1/NOS2-transfected ST-1 cells
for 16 h in the presence of vehicle,
L-NAME, or the inactive
stereoisomer D-NAME. This
maneuver created "NO-deficient"
(L-NAME) and "NO-replete" (vehicle or D-NAME) conditions.
Cell viability, as indexed by trypan blue exclusion and cloning
efficiency, was not different between the NO-replete and NO-deficient
cells (data not shown), indicating that prolonged exposure to NO in
this system was not grossly cytotoxic. After the incubation period, the
cells were first assayed for ouabain-sensitive
86Rb+
uptake to assess
Na+-K+-ATPase
activity in the intact cell. As shown in Fig.
2,
pcDNA3.1/NOS2 ST-1 cells grown in the
presence of vehicle or D-NAME
exhibited rates of ouabain-sensitive
86Rb+
uptake that were ~35% lower than the cells grown in the presence of
L-NAME. Moreover, no
Sch-28080-sensitive component of
86Rb+
uptake was observed in the vehicle-treated (3,610 ± 123 vs. 3,420 ± 84 cpm · well1 · min
1,
control and Sch-28080-treated, respectively;
n = 4, P = NS) or
L-NAME-treated cells (4,797 ± 203 vs. 4,637 ± 114 cpm · well
1 · min
1,
control and Sch-28080-treated, respectively;
n = 4, P = NS). Therefore, the greater rate
of ouabain-sensitive
86Rb+
uptake in the L-NAME-treated
cells reflects enhanced
Na+-K+-ATPase
activity. In the aggregate, these data indicate that constitutive NO
production inhibited sodium pump activity or expression.
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Endogenous NO production inhibits expression of the
Na+-K+-ATPase
1-subunit gene.
Diminished rates of ouabain-sensitive
86Rb+
uptake could represent an inhibitory effect of NO on mitochondrial ATP
production, Na+ entry,
Na+-K+-ATPase
itself, or
Na+-K+-ATPase
gene expression. To determine whether the inhibitory effect of
endogenous NO on sodium pump function occurred at the level of
Na+-K+-ATPase
gene expression, we perfomed Northern analysis to compare steady-state
levels of
Na+-K+-ATPase
1-subunit mRNA in NOS2-transfected
cells grown in the presence of vehicle,
D-NAME, or
L-NAME. As shown
in Fig. 3,
A and
B, the abundance of
Na+-K+-ATPase
1-subunit mRNA in the cells exposed to vehicle or
D-NAME was ~40% less than in
the cells studied in the presence of
L-NAME. GAPDH mRNA levels were
not significantly different among the three groups, indicating that the
effect of NO to suppress
Na+-K+-ATPase
1-subunit gene expression was not generalized. Because the effect of
NO to limit Na+ entry could
conceivably downregulate the
Na+-K+-ATPase
1-subunit gene, we used amiloride and bumetanide to inhibit Na+ entry mediated by epithelial
Na+ channels,
Na+/H+
exchangers, and the
Na+-K+-2Cl+
cotransporter, and then measured the abundance of
Na+-K+-ATPase
1-subunit mRNA. As seen in Fig.
3C, incubation of
NOS2-transfected cells studied in the
presence of L-NAME together with
500 µM amiloride or 100 µM bumetanide for 16 h did not
significantly alter steady-state levels of
Na+-K+-ATPase
1-subunit mRNA. A similar lack of effect of amiloride and bumetanide
was observed in pcDNA3.1-transfected cells
(n = 3, data not shown). Therefore,
the inhibitory effect of NO on the expression of
Na+-K+-ATPase
1-subunit mRNA appears to be direct and unrelated to changes in
amiloride- or bumetanide-inhibitable
Na+ entry mechanisms.
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Endogenous NO production inhibits transcription of
the
Na+-K+-ATPase
1-subunit gene.
To determine whether the diminished expression of
Na+-K+-ATPase
1-subunit mRNA in the untreated
NOS2-transfected cells resulted from
lower rates of de novo gene transcription, we performed nuclear run-on
transcription assays using nuclei isolated from
NOS2-transfected cells treated with
vehicle, D-NAME, or
L-NAME for 16 h. The
NOS2-transfected cells treated with
vehicle or D-NAME exhibited significantly lower rates of Na+-K+-ATPase
1-subunit gene
transcription compared with those treated with
L-NAME (Fig.
4). In contrast, all three groups exhibited comparable rates of
-actin gene transcription (Fig. 4).
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DISCUSSION |
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The introduction of a functionally active
NOS2 gene into cultured ST-1 cells
allowed us to examine the effects of constitutive, high-output NO
production on epithelial cell function without the confounding effects
of LPS and cytokines needed to induce NOS2 expression, the limitations
of NO donors (4, 19), or vascular effects that might indirectly
modulate epithelial function. By using
L-NAME to control the operation
of NOS2 in these cells, we were able to explore, during a defined time
period, the effects of continuously produced NO on
Na+-K+-ATPase
expression and function. The concordance of our ouabain-sensitive 86Rb+
uptake, Northern blot, and nuclear run-off transcription data indicates
that the effect of NO in this model system is exerted principally on
the regulation of
Na+-K+-ATPase
1-subunit gene expression rather than directly on the enzyme itself.
Our results indicate that NO tonically produced by these cells
selectively suppresses
Na+-K+-ATPase
1-subunit gene transcription and, consequently, sodium pump
activity. This novel effect of NO may contribute to the known natriuretic and diuretic actions of NO in vivo, particularly under conditions in which the kidney is exposed to endogenous or exogenous inflammatory mediators known to provoke NOS2 expression.
The fact that expression of GAPDH (Fig. 3) and -actin (Fig. 4)
transcripts was not affected by NO under the experimental conditions
employed suggests that the inhibitory effect of NO on the
Na+-K+-ATPase
1-subunit gene does not result from a generalized suppression of the
transcriptional machinery of the cell, but rather involves mechanisms
that are necessary, and possibly specific, for transcription of the
Na+-K+-ATPase
1-subunit gene. In an in vivo model of NO excess, S. H. Liu and T. J. Sheu (26) reported reduced protein expression of
Na+-K+-ATPase
1-subunit, but not of
2- or
3-subunits, in the sciatic nerves
of rats treated with LPS. These data suggest that the inhibitory effect
of NO may be selective for the
Na+-K+-ATPase
1-subunit and may occur in other tissues. However, because LPS
regulates the expression of numerous genes, further studies are needed
to determine whether continuous NO exposure itself alters the
expression of the
Na+-K+-ATPase
2-,
3-, and
4-subunits. Additionally, the present study did
not specifically examine the effects of NO on other aspects of
Na+-K+-ATPase
bioregulation, which include pretranslational, translational, and
posttranslational controls of
- and
-subunit expression, as well
as controls of
-
-subunit assembly, subcellular distribution, and
phosphorylation state of the holoenzyme (5, 10).
Because blockade of Na+ entry
pathways with amiloride and bumetanide did not mimic the decrement in
the steady-state abundance of
Na+-K+-ATPase
1-subunit mRNA observed in the
NOS2-transfected cells treated with
L-NAME, the NO effect on
Na+-K+-ATPase
1-subunit transcription in the
NOS2-transfected ST-1 cells is largely
independent of, but perhaps additive to, physiological changes in
Na+ entry. It is noteworthy that
NO has been shown to inhibit
Na+/H+
exchange (40) and Na+-selective
cation channels (9, 13, 17) in other epithelial cell types. Because the
production of NO in our system was unregulated and, at micromolar
concentrations, exceeded the concentrations of NO typically used for
the signaling actions of the molecule, it is not known whether
NOS2-generated NO production might coordinately regulate
Na+ entry and
Na+-K+-ATPase
activity in response to changes in body sodium balance. The finding that NOS2 protein levels in the outer and inner medullae of
rats maintained on a high-NaCl diet were 50% greater than in these
renal zones of rats fed a low-NaCl diet (31) suggests such a possibility.
Our results parallel and extend those of Guzman and co-workers (14),
who studied
Na+-K+-ATPase
activity in mouse proximal tubule cells pretreated with LPS and
IFN-. These authors observed that
Na+-K+-ATPase
activity decreased beginning 4 h after LPS and IFN-
treatment, presumably reflecting the time needed for induction of NOS2 protein expression, but that maximal inhibition was achieved after 24 h. The
latter result suggests that the cumulative dose or duration of exposure
to NO was important. The magnitude of inhibition (34%) of
Na+-K+-ATPase
activity observed in their study is comparable to the ~35%
inhibition reported here. Because it was not determined whether Na+-K+-ATPase
1-protein or mRNA abundance were also reduced, it is not known
whether the effect observed in their studies was related to effects on
Na+-K+-ATPase
-subunit gene expression.
We did not identify any Sch-28080-sensitive component of
86Rb+
uptake in either naive or
NOS2-transfected ST-1 cells. These
results contrast with those of Younes-Ibrahim and co-workers (54), who detected Sch-28080-sensitive
K+-ATPase activity in
permeabilized MTALs microdissected from the rat. The reasons for this
apparent discrepancy are unclear, but may reflect species differences,
dedifferentiation of the ST-1 cells, or the fact that we measured
86Rb+
uptake in intact cells, whereas they measured enzymatic activity in
permeabilized tubules (i.e., it has not been established that the rat
MTAL expresses a Sch-28080-sensitive, active
K+ transport system in its plasma
membrane). Because immunoreactivity for the known
H+-K+-ATPase
-subunit isoforms has not been demonstrated in this nephron segment
(42, 52), the molecular identity of the transporter mediating the
Sch-28080-sensitive K+-ATPase
activity in permeabilized rat MTALs remains unknown. These contrasting
results highlight the need for caution in extrapolating the results
obtained in the immortalized ST-1 cell line to the MTAL in vivo.
Although ST-1 cells exhibit several of the phenotypic properties of the
MTAL in vivo, they do not perfectly model the MTAL of normal kidney.
For example, we previously detected in ST-1 cells expression of mRNA
encoding mBSC2 (also termed NKCC1) (25), the "secretory" isoform
of the bumetanide-sensitive Na-K-2Cl cotransporter that is not normally
expressed in the MTAL in vivo (12, 18). Because cell culture itself has
been shown to induce expression of BSC2 in proximal tubule and other
cells (39), the significance of this finding is unknown. In addition,
further studies will be needed to determine the specific Na-K-2Cl
cotransporter(s) that mediates the bumetanide-sensitive
86Rb+
uptake of ST-1 that we observed in the present study.
In normal rats, the MTAL basally expresses high NOS2 mRNA levels (1, 33, 35), NOS2 immunoreactivity (48), and strong NADPH diaphorase activity (32), a histochemical marker of NOS activity, suggesting that the NOS2 expressed in this segment may be constitutively active. In contrast, the MTAL of normal rat does not appear to express NOS1 or NOS3 (2). Why the MTAL, and not other nephron segments, might tonically express NOS2 has not been established. The MTAL is the major nephron site responsible for the concentration and dilution of the urine, and it accomplishes these tasks, in large part, using extremely high rates of active sodium reabsorption (11). The electrochemical gradients established and maintained by the Na+-K+-ATPase in the basolateral membrane of the MTAL serve as the principal driving force for this work. An inhibitory effect of NO on Na+-K+-ATPase gene expression in the MTAL would be expected to inhibit NaCl absorption by this segment and facilitate natriuresis if NaCl absorption were also inhibited in the cortical TAL (20); if NaCl absorption in the cortical TAL were not concomitantly reduced, the inhibitory effect of NO on MTAL NaCl absorption may only reduce the medullary osmolality and impair urinary concentration. Such direct tubular effects, however, may be difficult to discriminate from the effects of NO on the renal microcirculation (37).
Whereas the present study was designed to study the effects of sustained NO production and exposure on the Na+-K+-ATPase of an MTAL-derived cell line, others have demonstrated rapid effects of NO on K+ channels of the MTAL. Lu and co-workers (28, 29) measured the acute effects of NO donors and NOS inhibitors on the activity of the apical 70-pS K+ channel in cell-attached patches of the rat MTAL. They concluded that NO, via a cGMP-dependent mechanism, rapidly activates this K+ channel (28). Moreover, they found that high angiotensin II concentrations, through stimulation of endogenous NO production, activate the channel (29). That NO could both activate an apical K+ channel and inhibit the Na+-K+-ATPase in the MTAL is perplexing, if indeed these actions occur simultaneously. Further studies to determine whether ST-1 cells express an apical K+ channel comparable to that of rat MTAL and to examine activity of the apical channel after more prolonged exposure to NO would help to clarify this issue.
We speculate that the inhibitory effect of NO on
Na+-K+-ATPase
1-subunit transcription is related to interference with DNA-protein interactions important for transcription of the
Na+-K+-ATPase
1-subunit gene. Several cis
elements and trans factors have been
implicated in transcriptional control of the
Na+-K+-ATPase
1-subunit gene (21, 51), and NO has been shown to modulate the
binding or activity of several transcription factors in other cell
types (7, 27, 38, 43, 47). Given the ubiquity of the
Na+-K+-ATPase
and its importance to the function of all eukaryotic cells, and the
ubiquity of NOS2 in immune-stimulated mammalian cell types, this novel
action of NO may have broad physiological and pathophysiological implications.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-47981 and DK-50745 (to B. C. Kone), and was completed during the tenure of B. C. Kone as an Established Investigator of the American Heart Association.
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FOOTNOTES |
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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.
1 We were concerned that release of reactive oxygen species, such as superoxide anion and hydroxyl radical, from the large number of dying cells (those not successfully incorporating the transfected neomycin resistance gene) might react with NO from the successfully transfected cells to produce toxic metabolites that would limit clonal proliferation. However, we did not formally test whether inclusion of L-NAME in the culture medium during G418 selection was required for generation of the NOS2-expressing clones.
Address for reprint requests and other correspondence: B. C. Kone, Depts. of Internal Medicine and Integrative Biology, Pharmacology, and Physiology, Univ. of Texas Medical School at Houston, 6431 Fannin, MSB 4.148, Houston, TX 77030 (E-mail: bkone{at}heart.med.uth.tmc.edu).
Received 15 July 1998; accepted in final form 31 December 1998.
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