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: gardner@itsa.ucsf.edu

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. 

  10.1074/jbc.M111117200Access the most updated version of this article at doi:

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