osmoregulation of natriuretic peptide ...2001/12/14 · natriuretic peptide receptor (npr-a) plays...
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OSMOREGULATION OF NATRIURETIC PEPTIDE RECEPTOR SIGNALING IN INNER MEDULLARY COLLECTING DUCT. A REQUIREMENT FOR P38
MAPK.
Songcang Chen and David G. Gardner*
Diabetes Center/Metabolic Research Unit and *Department of Medicine University of California at San Francisco
San Francisco, CA
Address correspondence to: David G. Gardner, M.D.
Diabetes Center/ Metabolic Research Unit 1109 HSW
University of California at San Francisco San Francisco, CA 94143-0540
Tel: (415) 476-2729 FAX: (415) 476-1660
Email: [email protected]
Running Title: Osmoregulation of natriuretic peptide receptor
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on December 14, 2001 as Manuscript M111117200 by guest on O
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ABSTRACT
In the inner medullary collecting duct of the terminal nephron, the type A
natriuretic peptide receptor (NPR-A) plays a major role in determining urinary sodium
content. This nephron segment, by virtue of its medullary location, is subject to very
high levels of extracellular tonicity. We have examined the ability of medium tonicity to
regulate the activity and expression of this receptor in cultured rat inner medullary
collecting duct cells. We found that NaCl (75 mM) and sucrose (150 mM), but not urea
(150 mM) increased natriuretic peptide receptor activity, gene expression and promoter
activity. The osmotic stimulus also activated extracellular signal regulated kinase (ERK),
c-Jun amino terminal kinase (JNK) and p38 mitogen activated protein kinase (p38
MAPK). In the latter instance the β isoform was selectively activated. Inhibition of p38
MAPK with SB203580 blocked the osmotic induction of receptor activity and
expression, as well as receptor gene promoter activity, while inhibition of ERK with
PD98059 had no effect. Cotransfection of p38β MAPK together with the receptor gene
promoter resulted in amplification of the osmotic stimulation of the latter, while
cotransfection of dominant negative MKK6, but not dominant negative MEK, completely
blocked the osmotic induction of receptor promoter activity. Collectively, the data
indicate that extracellular osmolality stimulates receptor activity and receptor gene
expression through a specific p38β-dependent mechanism and raise the possibility that
changes in medullary tonicity could play an important role in the regulation of renal
sodium handling in the terminal nephron.
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INTRODUCTION
Atrial (ANP) and brain (BNP) natriuretic peptide exert potent natriuretic,
vasorelaxant and anti-mitogenic activity by virtue of their interaction with a specific, high
affinity, guanylyl cyclase-linked receptor called the type A natriuretic peptide receptor
(NPR-A) (1). These receptors are located on the surface of a variety of cells including
vascular endothelial and smooth muscle cells as well as epithelial cells lining the inner
medullary collecting duct (IMCD) where activation leads to an increase in intracellular
cyclic GMP levels, suppression of sodium transport on the luminal surface and a net
increase in urinary sodium excretion (2). The IMCD is involved in handling up to 5% of
total filtered sodium load and because of its location in the terminal segment of the
nephron, it plays a critical role in the determination of final urinary sodium concentration.
While we know a great deal about how the natriuretic receptors function (3), we
know very little about their regulation. NPR-A is acutely desensitized following
exposure to ANP (4) or phorbol ester (5), reflecting dephosphorylation of the receptor
protein. At the level of gene expression, the NPR-A gene has been shown to be regulated
by glucocorticoids (6), transforming growth factor-β (7) and chorionic gonadotrophin (8).
The NPR-A gene also undergoes homologous down regulation following exposure to its
natriuretic peptide ligand or the ligand’s second messenger (cGMP) (9) but the precise
mechanism(s) underlying this regulation remains undefined.
By virtue of its location in the inner medulla of the kidney, the IMCD is subject to
substantial variation in the level of extracellular tonicity. Though a number of studies
have been carried out investigating the osmoregulation of gene expression in various cell
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types including those of the IMCD (10-12), few have focused on genes involved in
sodium handling in this segment of the nephron . For this reason we have undertaken a
study to examine the osmoregulation of NPR-A activity and expression in primary
cultures of rat IMCD cells. We have found that the activity of NPR-A, the expression of
its gene and the activity of the NPR-A gene promoter are all stimulated by increased
extracellular tonicity. This stimulation appears to traffic selectively through the β
isoform of p38 MAPK.
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EXPERIMENTAL PROCEDURES
Materials. [α32P]-dCTP was purchased from Dupont/New England Nuclear Research
Products (Boston, MA). ANP was obtained from Phoenix Pharmaceuticals Inc
(Mountain View, CA). RNeasy mini kit was purchased from QIAGEN Inc. (Santa
Clara, CA). Primer-it® RMT kit, hybridization solution and Nuctrap push columns were
purchased from Stratagene Inc. (La Jolla, CA). Anti-ERK, anti-JNK, anti-phospho-p38,
anti-p38α and anti-p38β antibodies were obtained from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Other reagents were obtained through standard commercial suppliers.
Isolation and culture of IMCD cells. Animal studies were approved by the UCSF
Committee on Animal Research. Adult Sprague-Dawley rats were euthanized by CO2
narcosis followed by bilateral thoracotomy. Kidneys were excised and bivalved with a
scalpel blade. The inner medullary tissue was dissected free from the outer medulla,
minced into one cubic millimeter fragments and digested with 1 mg/ml collagenase at 37
C with gentle agitation during each 30 min cycle. IMCD cells were enriched in the
preparation using hypotonic lysis as described previously (13). The cells were
resuspended in medium-1 [1:1 mixture of Dulbecco’s Modified Eagles Medium (DMEM)
and Ham’s F-12 Medium supplemented with 10% fetal bovine serum (FBS), 42 mM
sodium bicarbonate, 100 IU/ml penicillin and 100 ug/ml streptomycin] and seeded on to
culture plates. After 24h, the cells were placed in K-1 medium [1:1 mixture of DMEM
and Ham’s F-12 medium supplemented with 10 mM HEPES (pH 7.4), 42 mM sodium
bicarbonate, 5 ug/ml insulin, 50 nM hydrocortisone, 5 ug/ml transferrin, 5 pM
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triiodothyronine, 100 IU/ml penicillin and 100 ug/ml streptomycin] and cultured for 3-4
days.
Measurement of ANP-stimulated cGMP levels. IMCD cells were grown to ~80%
confluence and incubated for different periods of time under conditions outlined in the
individual figure legends. For measurement of ANP-stimulated cGMP (ANP-s-cGMP)
accumulation, cells were washed three times with prewarmed (37 C) phosphate-buffered
saline (PBS) and incubated with 0.5 ml of DMEM containing 0.5 mM
isobutylmethylxanthine (IBMX) and 10 mM HEPES (pH 7.4) for 10 min at 37 C. 10-7 M
ANP was added to the medium and the incubation was continued for another 10 min.
The reaction was stopped by removal of medium and addition of 0.3 ml of 12%
trichloroacetic acid (TCA). The extraction was continued for 30 min at 4 C. The
contents of the plate were collected and centrifuged to pellet particulate material; the
supernatant fraction was extracted four times with 0.5 ml of water-saturated ether. cGMP
levels were determined by radiommunoassay, after acetylation of the sample and
standard, using a commercial 125I-cGMP RIA kit (NENTMLlife Science, Boston, MA)
RNA isolation and Northern blot analysis. IMCD cells were plated in 10-cm dishes,
cultured and treated with different reagents as indicated in the individual figure legends.
Total RNA was extracted from cells using the RNeasy minikit according to instructions
provided by the manufacturer. Total RNA was denatured and separated on a gel
containing 2.2% formaldehyde, transferred to a nitrocellulose filter and hybridized to
radiolabeled cDNA probe as described previously (14). A 1.2 kb EcoR I fragment of the
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rat NPR-A cDNA was isolated from vector sequence, radiolabeled using the primer-itR©
RMT kit (Stratagene) and separated from free nucleotide using Nuctrap push columns
(Stratagene). Membranes were prehybridized for 30 min at 68 C and hybridized with
32P-labeled cDNA for 1 h at 68 C in hybridization solution provided by Stratagene. All
membranes were subsequently stripped and rehybridized with a radiolabeled 1150 bp
Bam HI/EcoR I fragment of 18S rDNA to permit normalization among samples for
differences in RNA loading and/or transfer to the filter. Hybridization signal was detected
by autoradiography and quantified using the National Institutes of Health (NIH) Image
program.
Plasmid constructions. The –1575 rat NPR-A promoter fragment was originally
isolated as a Bgl II (5’ terminus)/Nar I (3’ terminus) fragment and cloned in the pFoxLuc
vector (9). Subsequent studies suggested that pFoxLuc contains cryptic transcriptional
regulatory elements that idiosyncratically responded to selected experimental
perturbations including exposure to hypertonic medium. To circumvent potential
complications in interpretation of experimental data, the Bgl II/Nar I fragment was
recloned into the Bgl II/Nar I sites in PGL3-LUC. Subsequent analyses confirmed that
this vector was only modestly responsive to extracellular tonicity (see below). pcDNA3-
p38β and pcDNA3-p38β(AF), a kinase-defective mutant of p38β, were kindly provided
by Dr. Jiahuai Han of Scripps Research Institute (La Jolla, CA) (15). pcDNA3-
MKK6AL, a dominant-negative MKK6 mutant was provided by J. R. Woodgett
(University of Toronto, Toronto, Canada) (16). A eukaryotic expression vector encoding
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dominant-negative MEK (K97R) was provided by M . Karin (University of California,
San Diego) (17).
Transfection and luciferase Assay. Cells were plated in 6-well dishes and grown to
~70% confluence. At that time, transfection was carried out with Lipofectin Reagent
(Life Technologies) using a protocol recommended by the manufacturer.
One µg of -1575NPR-A-LUC with 0.2 µg CMV-β-galactosidase (β -gal) were
introduced into each well. The DNA-liposome suspension was incubated with the
cultures for 5-6 h at 37 C in Opti-MEMI Reduced Serum Medium (Gibco-BRL). The
suspension was then removed and replaced with K-1 medium for the ensuing 24 h, at
which point cells were treated with different concentrations of sucrose, NaCl or urea in
K1 medium for defined periods of time. At the end of the incubation, cells were washed
three times with PBS and lysed with Promega lysis buffer. Luciferase activity was
measured using the Luciferase Assay System (Promega). β-galactosidase activity was
assayed using the Galactolight Plus Chemiluminescence Assay (Tropix, Bedford, MA).
Luciferase measurements were normalized for β-galactosidase activity in the individual
cultures.
Immunoprecipitation and kinase assays. Cells were washed twice with PBS and
scraped into 0.8 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton
X-100, 5 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM sodium orthovanadate, 10 mM
sodium fluoride, 1 mM glycerophosphate, protease inhibitor cocktail tablet (50 ml lysis
buffer /tablet; Boehringer Mannheim, GmbH, Germany). The lysates were clarified by
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centrifugation in a benchtop centrifuge at 15,000 rpm for 10 min at 4°C. 200 µg of
supernatant protein was incubated with 1 µg of anti-ERK2, anti-p38α, anti-p38β or anti-
JNK1 antibody and 10 µl of protein G-Sepharose for 1-2 h at 4° C. Immunoprecipitates
were washed three times with lysis buffer and once with kinase buffer (25 mM HEPES,
pH 7.4, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT) without ATP. After washing, the
immunoprecipitates were resuspended in 30 µl of kinase buffer containing 20 µM ATP,
10 µCi [γ-32P]ATP and 10 µg myelin basic protein (MBP) for measurements of
extracellular signal-regulated protein kinase (ERK), p38α and p38β activities, or 5 µg
glutathione S-transferase-c-Jun (GST-c-JUN) for c-Jun NH2-terminal kinase (JNK)
activity, and incubated at 30°C for 20 min. The incubation mixtures were electrophoresed
on 15% SDS-polyacrylamide gels, which were then dried and exposed to x-ray film.
Phosphorylation of each substrate protein was quantitated by Scion Image.
Immunoblot analysis. Cell lysates (30 µg soluble protein) was subjected to 12.5% SDS-
PAGE and transferred onto nitrocellulose membranes (Amersham Chemical Corp.,
Amersham, IL). The membranes were blocked with 5% nonfat milk in 50 mM Tris-HCl,
pH 7.5, 150 mM NaCl, 0.1% Tween 20 (TBST) and probed with anti-phospho-p38
antibody. A horseradish peroxidase-conjugated second antibody was employed to detect
immunoreactive bands using the enhanced chemiluminescence (ECL) Western blotting
detection system (Amersham Chemical Corp.).
Statistical analysis. Data were evaluated using one-way ANOVA with Newman-Keuls
test for significance.
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RESULTS
Osmoregulation of NPR-A activity and gene expression
Activity of NPR-A in rat IMCD cells, assessed as ANP-sensitive cyclic GMP
(ANP-s-cGMP) generation, proved to be very sensitive to extracellular osmolality. As
shown in Fig. 1A, both sodium chloride as well as sucrose effected significant increments
in ANP-sensitive cGMP generation in these cultures while addition of the osmotically
inert solute urea to the culture medium was devoid of activity. Addition of 150 mM
sucrose (final calculated tonicity in the medium = 404 mosm/kg H2O) or 75 mM NaCl
increased NPR-A activity almost 3 fold over a 24 hr incubation period. Equi-osmolar
(150 mM) urea was without effect over the same 24 hr period.
This increase in NPR-A activity was accompanied by an increase in NPR-A gene
expression. As shown in Fig. 1B, there was a time-dependent increase in NPR-A
transcript levels following exposure to either sucrose (150 mM) or NaCl (75 mM) which
was noted as early as 4 hrs and reached a maximum, without peaking, after 24 hrs. This
increase in NPR-A mRNA levels was related, at least in part, to increased transcription of
the NPR-A gene. Transient transfection of a chimeric NPR-A promoter-luciferase
reporter into IMCD cells (Fig. 1C) revealed a sucrose- and NaCl-dependent induction of
promoter activity which began as early as 4 hrs and reached a maximum after 24 hrs of
incubation. The background vector (pGL3-LUC) was affected minimally by addition of
either sucrose or NaCl (fold induction relative to control: 0.98 +/- 0.12 at 4 hrs, 1.38 +/-
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0.2; mean +/- SD, n=3). Osmotically inactive urea had no effect on either NPR-A gene
transcript levels or NPR-A promoter activity.
Osmotic stimulation of MAPK activity in IMCD cells.
Members of the extended mitogen-activated protein kinase (MAPK) family are
known to be regulated by a variety of biochemical and physical stimuli including, in
selected cases, extracellular tonicity (18-22). We examined the ability of NaCl (75 mM)
to stimulate ERK, JNK and p38 MAPK in cultured rat IMCD cells. As shown in Fig. 2A,
all three kinases were activated by the osmotic stimulus. ERK was stimulated to the
greatest degree (almost 5 fold) 20 min following application of the osmotic stimulus.
JNK stimulation followed a similar time course (maximal at 20 min) but reached a
maximal induction of only 2.5 fold over control. p38 MAPK activation, assessed through
measurements of phospho-p38 MAPK levels in IMCD cell extracts, peaked slightly later
(60 min) with maximal induction of ~3 fold. As expected the ERK induction by NaCl
was completed abrogated by the MEK (ERK kinase) antagonist PD98059, as was the p38
MAPK induction by SB203580 (Fig. 2B). SB203580, at the concentrations employed
here, had no effect on JNK activity (Fig. 2B).
p38 MAPK exists as two major isoforms, p38 MAPK α and β. Importantly, these
isoforms can differentially activate independent downstream targets (15). Both isoforms
exist in the rat IMCD cell (Fig. 3). Interestingly, it appears to be the β- rather than the α-
isoform that is induced by extracellular tonicity. As shown in Fig. 3A, the β-isoform of
p38 MAPK was induced ~2.5 fold by NaCl (75 mM) while the α-isoform was induced
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little, if at all. Similar to the finding with phosp-p38 MAPK in Fig. 2, the activity of p38
MAPK β was readily suppressed by SB203580 (Fig. 3B).
Inhibition of p38 MAPK blocks osmo-stimulation of NPR-A.
Treatment of IMCD cells with the p38 inhibitor SB203580 almost completely
blocked the NaCl-dependent (Fig. 4A) and sucrose-dependent (Fig. 4B) stimulation of
NPR-A activity, assessed as ANP-dependent cyclic GMP synthesis. The MEK inhibitor
PD98059 had no effect on NaCl-dependent activation of NPR-A, and neither inhibitor
had any effect on basal activity. This effect on NPR-A activity was mirrored at the level
of NPR-A gene expression. As shown in Fig. 5, pre-treatment with SB203580, but not
PD98059, led to a significant reduction in the osmotic stimulation of NPR-A mRNA
levels in IMCD cells. The latter effect was accompanied by a reduction in NPR-A gene
transcriptional activity. SB203580 (Fig. 6A), but not PD98059 (Fig. 6B), effected a
dose-dependent reduction in NPR-A gene promoter activity following transient
transfection of -1150 NPR-A-luciferase into IMCD cells.
To examine the p38-dependence of the osmotic induction more closely we
employed a genetic approach. Cotransfection of an expression vector encoding the β
isoform of p38 MAPK together with the NPR-A-luciferase reporter resulted in a 2-fold
increment in promoter activity, slightly less than that seen with NaCl (Fig. 7A).
Cotransfection of a vector encoding the beta isoform mutated at the kinase active site
(p38-β AF) resulted in no net increment in promoter activity. Of note, cotranfection of
p38-β into cells that were subsequently exposed to NaCl resulted in a greater-than-
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additive increment in reporter activity implying a synergistic interaction between the
osmotic stimulus and levels of p38β-MAPK in the IMCD cell. This interaction was not
shared with the p38-β AF mutant. Cotransfection of a dominant negative mutant of
MKK6, a p38 MAPK kinase which targets both the α and β isoforms of the kinase,
resulted in a dose-dependent reduction in NaCl-dependent NPR-A promoter activation
while dominant negative MEK (ERK kinase) was without effect (Fig. 7B). Neither
mutant affected basal promoter activity (i.e. in the absence of the NaCl induction) to a
significant degree.
DISCUSSION
This study provides the first evidence for regulation of natriuretic peptide receptor
gene transcription by extracellular tonicity. It also suggests that this occurs, in large part,
through a mechanism operating downstream of p38β MAPK activation.
The role of p38 MAPK in signaling osmotic activity has been demonstrated in a
number of other systems including yeast, where the p38 MAPK homologue HOG1 plays
a critical role in signaling an increase in glycerol-3-phoshate dehydrogenase gene
expression following exposure to an osmotic stimulus (23). Activation of p38 MAPK has
also been linked to the osmotic induction of aldose reductase (22), betaine/γ-amino-n-
butyric acid (GABA) transporter (BGT-1) (24), sodium-dependent myoinositol
transporter (SMIT) (24) and the serum-and glucocorticoid-inducible protein kinase (Sgk)
(21). The selective activation of p38β by increased extracellular tonicity demonstrated
here is unique. The α and β isoforms of p38 MAPK are often expressed in the same cell
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type and may be regulated in parallel. It is noteworthy, however, that they have the
capacity to act on different substrates (15), a property which could conceivably result in
considerable differences in biological activity. For example, activation of p38β results in
amplification of the hypertrophic response when overexpressed in neonatal rat cardiac
myocytes while activation of the α isoform leads to an increase in programmed cell death
(25). Preferential signaling of the osmotic stimulus through the β isoform in IMCD cells
suggests that independent regulatory circuitry may govern the activity of these two
isoforms in this nephron segment. Thus, as in the cardiac myocyte, it is conceivable that
differential activation of these two isoforms in the IMCD could result in opposing
physiological activities under selected conditions.
It is noteworthy that treatment of our cultures with SB203580 resulted not only in
reduction in the endpoint under examination (e.g. NPR-A mRNA levels or NPR-A
promoter activity) but a reduction in levels of phospho-p38 MAPK as well. Since
SB203580 blocks the activity of p38 itself, it is not immediately clear why it should exert
an effect on the activation/phosphorylation of the kinase. A similar result has been
reported with several other systems (26-28). This led to the suggestion that SB203580
may block the biological activity of p38 MAPK by binding to the inactive form of the
kinase and reducing its rate of activation (28), although a subsequent study seems to
refute this hypothesis (29). One potential explanation for this seemingly paradoxical
finding may be found in the structure of the p38 MAPK-SB203580 complex (30). Much
as one might predict, the inhibitor binds tightly in the ATP-binding pocket of the kinase
providing an obvious mechanism for the reduction in kinase activity. However, one
segment of the inhibitor projects toward the region harboring the tyrosine residue (Tyr182)
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that is phosphorylated as a prelude to kinase activation (31). This portion of the inhibitor
could conceivably result in an alteration in the three dimensional conformation of the
region surrounding this potential phosphorylation site resulting in a decrease in
phosphorylation of the kinase and impairment in its activation.
JNK and ERK activity are known to be activated by osmotic stimuli and, in some
cases, these activations have been tied to specific downstream effects (20,22). In the case
of induction of the NPR-A gene, neither JNK nor ERK appears to play a significant role.
The MEK inhibitor PD98059 completely blocked ERK activity but had no effect on
NPR-A gene promoter activity. SB203580, the p38 MAPK antagonist used in these
studies, has also been shown to inhibit JNK activity when used at higher concentrations
(>5x10-6M) (32). SB203580 completely suppressed both p38 MAPK activity and NPR-
A gene promoter activity but, at the concentrations employed in our studies, had no effect
on JNK activity. Thus, the data collectively support a role for a p38 MAPK, but not for
JNK or ERK, in trafficking the osmotic induction of NPR-A.
Previous studies have shown NPR-A-dependent guanylyl cyclase activity to be
either activated (33) or inhibited (34,35) by increases in extracellular tonicity. While none
of these studies was definitive in terms of elucidating the mechanisms involved, the
available data seems to suggest that activation dominates with longer exposure (i.e. ~24
hrs) and inhibition with short term exposure (i.e. minutes) to the osmotic stimulus. Our
findings support this model in that all of our experiments were carried out at longer time
intervals to maximize the probability of detecting changes in gene expression. The
mechanism(s) underlying the transient reduction of NPR-A activity at shorter time
intervals remains, as yet, undefined.
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Positioned at the most terminal portion of the nephron, the IMCD deals with up to
5% of filtered sodium load and is responsible for the final decision regarding urinary
sodium concentration. A variety of local and systemic regulatory factors act at the level
of the IMCD to influence sodium conservation in either a positive or negative fashion (2).
Thus, even modest perturbations in NPR-A activity in this nephron segment might be
predicted to result in significant changes in urinary sodium excretion. The IMCD, by
virtue of its position deep in the renal medulla, is also subject to tremendous variations in
extracellular tonicity. The osmotic regulation of NPR-A activity/expression
demonstrated here implies the existence of an additional local mechanism for regulating
sodium handling (i.e. through the natriuretic peptide/NPR-A system) in this critical
nephron segment that could play a significant role in the regulation of sodium balance.
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ACKNOWLEDGEMENTS
The authors would like to acknowledge the contributions of Dr. Li Cao to the early
phases of this work. We are also grateful to Dr. D. Garbers for plasmid reagents.
Supported by NIH grant HL45637.
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FIGURE LEGENDS
Fig. 1. Effect of increased osmolality on NPR-A activity, gene expression and promoter
activity in IMCD cells. (A) IMCD cells were cultured in DMEM (calculated osmolality
330 mosm/kg) containing 150 mM sucrose, 75 mM NaCl or 150 mM urea (these same
concentrations were used for all subsequent experiments) for different time intervals.
Osmolalities provided in the figure represent additions above that attributed to DMEM
itself. ANP-stimulated cGMP (ANP-s-cGMP) was determined as described in Materials
and Methods. Pooled data from 3-4 independent experiments are shown. Control cGMP
levels were 152 +/- 15 pmol/mg soluble protein. (B) IMCD cells at approximately 80%
confluence were incubated in medium containing sucrose, NaCl or urea for the times
indicated. RNA was extracted, size fractionated on agarose gels and blot-hybridized with
radiolabeled NPR-A cDNA or 18S rDNA probes as described in Materials and Methods.
Representative autoradiographs are shown and pooled data (n=4) are presented as
normalized NPR-A mRNA/18S rRNA ratios. (C) Cells were transfected with 1 µg –
1575 NPR-A LUC and 0.2 µg RSV-β-galactosidase as described in Materials and
Methods. Cells were treated with sucrose, NaCl or urea for indicated time intervals.
Cellular lysates were prepared and assayed for luciferase and β -galactosidase activities.
Luciferase activity was normalized for β -galactosidase expression. Data shown were
pooled from four independent experiments. *P<0.05, **P<0.01 vs. untreated group.
Fig. 2. Osmotic induction of MAPKs in IMCD cells. (A) Cells were cultured
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in the medium containing NaCl for indicated time intervals. Cells were lysed; lysates
were size-fractionated by SDS-PAGE and transferred onto membrances which were
probed with anti-phospho-p38 antibody. Immunoreactive bands were detected using ECL
system. In separate experiments, cellular lysates were immunoprecipitated with anti-
ERK2 or anti-JNK1 antibody. ERK and JNK activities were measured in an in vitro
kinase assay using MBP and GST-c-JUN as substrates, respectively. γ-32P labeled
products were then separated by SDS/PAGE. Representative experiments are shown.
Pooled data were derived from 3-4 experiments. (B) The cultures were pretreated with
10-5 M SB203580 or PD98059 for 1h and then treated with NaCl for 20 min. Activities of
p38, ERK and JNK were measured as described above. Representative experiments are
presented. Pooled data were obtained from four independent experiments. *P<0.05,
**P<0.01 vs. untreated group.
Fig. 3. NaCl stimulates p38 β activity but not p38α activity in IMCD cells. (A) Cultured
IMCD cells were treated with NaCl for different time intervals. Cells were lysed and
specific kinases were immunoprecipitataed with anti-p38α or anti-p38β antibody.
Activities were measured using MBP as substrate. Representative study and data derived
from 4-6 independent experiments are presented. (B) Cells were pre-ncubated with 10-5
M SB203580 for 1h and then treated with NaCl for 20 min. Cellular lysates were
immunoprecipitated with anti-p38 β antibody. p38 β activity was measured as described
above. Pooled data are from 3 independent experiments are shown. **P<0.01 vs.
untreated group.
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Fig. 4. p38 inhibitor, SB203580, suppresses NaCl- and sucrose-induced ANP-s-cGMP
in IMCD cells. (A) IMCD cells were grown to ~80% confluence, preincubated with 10-5
M PD203580 or SB203580 for 1h and then incubated with or without NaCl for 24h.
ANP-s-cGMP was determined as described in Materials and Methods. Pooled data from
3-4 independent experiments are shown. (B) Cells were pretreated with 10-5 M
SB203580 for 1h and then incubated with or without sucrose for 24h. ANP-s-cGMP
levels were determined by radiommunoassay. Bar graph is derived from three
independent experiments. Control cGMP levels were 158 +/- 18 pmol/mg soluble
protein. **P<0.01 vs. untreated group.
Fig. 5. Effect of PD98059 or SB203580 on NaCl-stimulated NPR-A mRNA levels in
IMCD cells. The cells were pretreated and treated as described as Fig. 4A. Total RNA
was prepared, size-fractionated on 2% agarose gels containing formaldehyde, transferred
on to nitrocellulose membranes and hybridized with α-32P labeled NPR-A cDNA or 18S
rDNA probes. Representative autoradiographs are shown. Pooled data (n=3) are
presented as normalized NPR-A mRNA/18S rRNA ratios. **P<0.01 vs. untreated group.
Fig. 6. Effect of PD98059 or SB203580 on NaCl-stimulated NPR-A-LUC in IMCD
cells. (A) Cells were transfected with 1 µg -1575 NPR-A LUC and 0.2 µg RSV-β-
galactosidase as described in Material and Methods. Cells were pretreated with the
indicated concentrations of SB203580 for 1 h and then treated with NaCl, in the
presence or absence of SB203580, for 24h. Cell lysates were assayed for luciferase and β
-galactosidase activities. Luciferase activity was normalized for β -galactosidase
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expression. Data shown were pooled from four independent experiments. (B) After
transfection with NPR-A-LUC and RSV- β -galactosidase, the cells were preincubated
with different concentration of PD203580 for 1h and then incubated with NaCl, in the
presence or absence of PD203580, for 24h. Luciferase activity was normalized for β -
galactosidase levels in individual samples. Pooled data (n=3) are presented. **P<0.01 vs.
untreated group.
Fig. 7. Effect of dominant-negative MEK and MKK6, and wild type p38 β on basal and
NaCl-stimulated NPR-A-LUC. (A) Cells were co-transfected with 1 µg –1575 NPR-A
LUC, 0.2 µg RSV- β -galactosidase and 1 µg p38 β or p38 β (AF). Twenty four hours
following transfection, cells were exposed to NaCl or vehicle for an additional 24h.
Luciferase activity was assayed as described above. Pooled data (n=3-4) are shown. (B)
Cells were co-transfected with 1 µg -1575 NPR-A LUC, 0.2 µg RSV- β -galactosidase
and different concentrations of dominant-negative MEK and MKK6 as indicated.
Twenty-four hours following transfection, cells were treated with or without NaCl for
24h. Data are presented from 4-5 separate experiments. **P<0.01 vs. untreated group.
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Songcang Chen and David G. Gardnerduct. A requirement for P38 MAPK
Osmoregulation of natriuretic peptide receptor signaling in inner medullary collecting
published online December 14, 2001J. Biol. Chem.
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