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J. Biol. Chem., Vol. 277, Issue 8, 6037-6043, February 22, 2002

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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^§¶

From the  Diabetes Center/Metabolic Research Unit and ^§ Department of Medicine, University of California at San Francisco, San Francisco, California 94143

Received for publication, November 20, 2001, and in revised form, December 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 NH₂-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK). In the latter instance the  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, whereas inhibition of ERK with PD98059 had no effect. Cotransfection of p38 MAPK together with the receptor gene promoter resulted in amplification of the osmotic stimulation of the latter, whereas 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 p38-dependent mechanism, raising the possibility that changes in medullary tonicity could play an important role in the regulation of renal sodium handling in the terminal nephron.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Atrial (ANP)¹ and brain 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.

Although 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- (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. Although a number of studies have been carried out investigating the osmoregulation of gene expression in various cell 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  isoform of p38 MAPK.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [-³²P]dCTP was purchased from PerkinElmer Life Sciences. ANP was obtained from Phoenix Pharmaceuticals Inc. (Mountain View, CA). RNeasy Minikit 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-p38, and anti-p38 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 University of California at San Francisco 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 1-mm² 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 Eagle's medium (DMEM) and Ham's F-12 medium supplemented with 10% fetal bovine serum, 42 mM sodium bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin) and seeded on to culture plates. After 24 h, 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 µg/ml insulin, 50 nM hydrocortisone, 5 µg/ml transferrin, 5 pM triiodothyronine, 100 IU/ml penicillin, and 100 µg/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 and incubated with 0.5 ml of DMEM containing 0.5 mM isobutylmethylxanthine and 10 mM HEPES (pH 7.4) for 10 min at 37 °C. 10⁷ M ANP was added to the medium, and the incubation was continued for another 10 min. The reaction was stopped by removing the medium and adding 0.3 ml of 12% trichloroacetic acid. 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 radioimmunoassay after acetylation of the sample and standard using a commercial ¹²⁵I-cGMP radioimmunoassay kit (PerkinElmer Life Sciences).

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 EcoRI fragment of the rat NPR-A cDNA was isolated from vector sequence, radiolabeled using the primer-it® 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 BamHI-EcoRI fragment of 18 S 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 NIH Image program.

Plasmid Constructions-- The 1575 rat NPR-A promoter fragment was originally isolated as a BglII (5' terminus)-NarI (3' terminus) fragment and cloned in the pFoxLuc vector (9). Subsequent studies suggested that pFoxLuc contains cryptic transcriptional regulatory elements that idiosyncratically respond to selected experimental perturbations including exposure to hypertonic medium. To circumvent potential complications in interpretation of experimental data, the BglII-NarI fragment was recloned into the BglII-NarI sites in PGL3-LUC. Subsequent analyses confirmed that this vector was only modestly responsive to extracellular tonicity (see below). pcDNA3-p38 and pcDNA3-p38(AF), a kinase-defective mutant of p38, 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, Canada) (16). A eukaryotic expression vector encoding 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 (Invitrogen) using a protocol recommended by the manufacturer.

One µg of 1575NPR-A-LUC with 0.2 µg CMV--galactosidase (-gal) were introduced into each well. The DNA-liposome suspension was incubated with the cultures for 5-6 h at 37 °C in Opti-MEM I reduced serum medium (Invitrogen). 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 K-1 medium for defined periods of time. At the end of the incubation, cells were washed three times with phosphate-buffered saline and lysed with Promega lysis buffer. Luciferase activity was measured using the Luciferase assay system (Promega). -Galactosidase activity was assayed using the Galactolight Plus chemiluminescence assay (Tropix, Bedford, MA). Luciferase measurements were normalized for -galactosidase activity in the individual cultures.

Immunoprecipitation and Kinase Assays-- Cells were washed twice with phosphate-buffered saline 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 mixture tablet (50 ml lysis buffer/tablet; Roche Molecular Biochemicals). The lysates were clarified by 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-p38, anti-p38, 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 dithiothreitol) without ATP. After being washing, the immunoprecipitates were resuspended in 30 µl of kinase buffer containing 20 µM ATP, 10 µCi of [-³²P]ATP, and 10 µg of myelin basic protein for measurements of extracellular signal-regulated protein kinase (ERK), p38, and p38 activities, or 5 µg of 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 of soluble protein) was subjected to 12.5% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences Inc.). The membranes were blocked with 5% nonfat milk in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20 and probed with anti-phospho-p38 antibody. A horseradish peroxidase-conjugated second antibody was employed to detect immunoreactive bands using the enhanced chemiluminescence Western blotting detection system (ECL, Amersham Biosciences Inc.).

Statistical Analysis-- Data were evaluated using one-way analysis of variance with Newman-Keuls test for significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Osmoregulation of NPR-A Activity and Gene Expression-- Activity of NPR-A in rat IMCD cells, assessed as ANP-stimulated cyclic GMP (ANP-s-cGMP) generation, proved to be very sensitive to extracellular osmolality. As shown in Fig. 1A, both sodium chloride and sucrose effected significant increments in ANP-stimulated cGMP generation in these cultures, whereas the osmotically inert solute urea added to the culture medium was devoid of activity. The 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-h incubation period. Equiosmolar (150 mM) urea was without effect over the same 24-h period.

View larger version (25K): [in this window] [in a new window]   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 under "Experimental Procedures." Pooled data from 3-4 independent experiments are shown. Control cGMP levels were 152 ± 15 pmol/mg soluble protein. B, IMCD cells at ~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 18 S rDNA probes as described under "Experimental Procedures." Representative autoradiographs are shown, and pooled data (n = 4) are presented as normalized NPR-A mRNA/18 S rRNA ratios. C, cells were transfected with 1 µg of 1575 NPR-A LUC and 0.2 µg of RSV--galactosidase as described under "Experimental Procedures." Cells were treated with sucrose, NaCl, or urea for the indicated time intervals. Cellular lysates were prepared and assayed for luciferase and -galactosidase activities. Luciferase activity was normalized for -galactosidase expression. Data shown were pooled from four independent experiments. *, p < 0.05; **, p < 0.01 versus untreated group.

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 h and reached a maximum, without peaking, after 24 h. 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 h and reached a maximum after 24 h of incubation. The background vector (pGL3-LUC) was affected minimally by the addition of either sucrose or NaCl (-fold induction relative to control: 0.98 ± 0.12 at 4 h, 1.38 ± 0.2; mean ± S.D., 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).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Osmotic induction of MAPKs in IMCD cells. A, cells were cultured in the medium containing NaCl for the indicated time intervals. Cells were lysed, and lysates were size-fractionated by SDS-PAGE and transferred onto membranes, which were probed with anti-phospho-p38 antibody. Immunoreactive bands were detected using an 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 myelin basic protein and GST-c-JUN as substrates, respectively. -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 105 M SB203580 or PD98059 for 1 h and then treated with NaCl for 20 min. Activity levels of p38, ERK, and JNK were measured as described above. Representative experiments are presented, and pooled data were obtained from four independent experiments. *, p < 0.05; **, p < 0.01 versus untreated group.

p38 MAPK exists as two major isoforms, p38 MAPK  and . 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  rather than the  isoform that is induced by extracellular tonicity. As shown in Fig. 3A, the  isoform of p38 MAPK was induced ~2.5-fold by NaCl (75 mM), whereas the  isoform was induced little, if at all. Similar to the finding with phospho-p38 MAPK in Fig. 2, the activity of p38 MAPK  was readily suppressed by SB203580 (Fig. 3B).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   NaCl stimulates p38 activity but not p38 activity in IMCD cells. A, cultured IMCD cells were treated with NaCl for different time intervals. Cells were lysed and specific kinases were immunoprecipitated with anti-p38 or anti-p38 antibody. Activities were measured using myelin basic protein as substrate. Representative studies and data derived from 4-6 independent experiments are presented. B, cells were preincubated with 105 M SB203580 for 1 h and then treated with NaCl for 20 min. Cellular lysates were immunoprecipitated with anti-p38 antibody. p38 activity was measured as described above. Pooled data from three independent experiments are shown. *, p < 0.05; **, p < 0.01 versus untreated group.

Inhibition of p38 MAPK Blocks Osmostimulation 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, pretreatment 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.

View larger version (9K): [in this window] [in a new window]   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 105 M PD203580 or SB203580 for 1 h, and then incubated with or without NaCl for 24 h. ANP-s-cGMP was determined as described under "Experimental Procedures." Pooled data from 3-4 independent experiments are shown. B, cells were pretreated with 105 M SB203580 for 1 h and then incubated with or without sucrose for 24 h. ANP-s-cGMP levels were determined by radioimmunoassay. The bar graph is derived from three independent experiments. Control cGMP levels were 158 ± 18 pmol/mg soluble protein. **, p < 0.01 versus untreated group.

View larger version (23K): [in this window] [in a new window]   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 for Fig. 4A. Total RNA was prepared, size-fractionated on 1% agarose gels containing formaldehyde, transferred onto nitrocellulose membranes, and hybridized with -32P-labeled NPR-A cDNA or 18 S rDNA probes. Representative autoradiographs are shown. Pooled data (n = 3) are presented as normalized NPR-A mRNA/18 S rRNA ratios. **, p < 0.01 versus untreated group.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of PD98059 or SB203580 on NaCl-stimulated NPR-A-LUC in IMCD cells. A, cells were transfected with 1 µg of 1575 NPR-A LUC and 0.2 µg of RSV--galactosidase as described under "Experimental Procedures." 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 24 h. Cell lysates were assayed for luciferase and -galactosidase activities. Luciferase activity was normalized for -galactosidase expression. Data shown were pooled from four independent experiments. B, after transfection with NPR-A-LUC and RSV--galactosidase, the cells were preincubated with different concentration of PD203580 for 1 h and then incubated with NaCl in the presence or absence of PD203580 for 24 h. Luciferase activity was normalized for -galactosidase levels in individual samples. Pooled data (n = 3) are presented. **, p < 0.01 versus untreated group.

To examine the p38 dependence of the osmotic induction more closely we employed a genetic approach. Cotransfection of an expression vector encoding the  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  isoform mutated at the kinase active site (p38(AF)) resulted in no net increment in promoter activity. Of note, cotransfection of p38- into cells that were subsequently exposed to NaCl resulted in a greater-than-additive increment in reporter activity, implying a synergistic interaction between the osmotic stimulus and levels of p38-MAPK in the IMCD cell. This interaction was not shared with the p38- AF mutant. Cotransfection of a dominant negative mutant of MKK6, a p38 MAPK kinase that targets both the  and  isoforms of the kinase, resulted in a dose-dependent reduction in NaCl-dependent NPR-A promoter activation, whereas 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.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of dominant-negative MEK and MKK6 and wild type p38 on basal and NaCl-stimulated NPR-A-LUC. A, cells were co-transfected with 1 µg of 1575 NPR-A LUC, 0.2 µg of RSV--galactosidase, and 1 µg of p38 or p38 (AF). 24 h after transfection, cells were exposed to NaCl or vehicle for an additional 24 h. Luciferase activity was assayed as described above. Pooled data (n = 3-4) are shown. B, cells were co-transfected with 1 µg of 1575 NPR-A LUC, 0.2 µg of RSV--galactosidase and different concentrations of dominant-negative MEK and MKK6 as indicated. 24 h after transfection, cells were treated with or without NaCl for 24 h. Data are presented from 4-5 separate experiments. **, p < 0.01 versus untreated group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 p38 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-phosphate 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/-amino-n-butyric acid transporter (24), sodium-dependent myoinositol transporter (24), and the serum- and glucocorticoid-inducible protein kinase (21). The selective activation of p38 by increased extracellular tonicity demonstrated here is unique. The  and  isoforms of p38 MAPK are often expressed in the same cell 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 p38 results in amplification of the hypertrophic response when overexpressed in neonatal rat cardiac myocytes, whereas activation of the  isoform leads to an increase in programmed cell death (25). Preferential signaling of the osmotic stimulus through the  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 end point 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) 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 (>5 × 10⁶ M) (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. Although none of these studies was definitive in terms of elucidating the mechanisms involved, the available data seem to suggest that activation dominates with longer exposure (i.e. ~24 h) 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.

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.

	ACKNOWLEDGEMENTS

We 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.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant HL45637.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. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: 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; E-mail: gardner@itsa.ucsf.edu.

Published, JBC Papers in Press, December 14, 2001, DOI 10.1074/jbc.M111117200

	ABBREVIATIONS

The abbreviations used are: ANP, atrial natriuretic peptide; ANP-s-cGMP, ANP-stimulated cyclic GMP; NPR-A, type A natriuretic peptide receptor; IMCD, inner medullary collecting duct; DMEM, Dulbecco's modified Eagle's medium; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun NH₂-terminal kinase; RSV, Rous sarcoma virus.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				D. G. Gardner, S. Chen, D. J. Glenn, and C. L. Grigsby Molecular Biology of the Natriuretic Peptide System: Implications for Physiology and Hypertension Hypertension, March 1, 2007; 49(3): 419 - 426. [Full Text] [PDF]

				S. Lee, Z. Wu, K. Sandberg, S-E. Yoo, and C. Maric Posttranscriptional mechanisms contribute to osmotic regulation of ANG type 1 receptors in cultured rat renomedullary interstitial cells Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R44 - R49. [Abstract] [Full Text] [PDF]

				A. R. Olzinski, T. A. McCafferty, S. Q. Zhao, D. J. Behm, M. E. Eybye, K. Maniscalco, R. Bentley, K. S. Frazier, C. M. Milliner, R. C. Mirabile, et al. Hypertensive target organ damage is attenuated by a p38 MAPK inhibitor: role of systemic blood pressure and endothelial protection Cardiovasc Res, April 1, 2005; 66(1): 170 - 178. [Abstract] [Full Text] [PDF]

				S. Chen, X.-P. Ni, M. H. Humphreys, and D. G. Gardner 1,25 Dihydroxyvitamin D Amplifies Type A Natriuretic Peptide Receptor Expression and Activity in Target Cells J. Am. Soc. Nephrol., February 1, 2005; 16(2): 329 - 339. [Abstract] [Full Text] [PDF]

				J. M. Capasso, C. J. Rivard, and T. Berl Synthesis of the Na-K-ATPase {gamma}-subunit is regulated at both the transcriptional and translational levels in IMCD3 cells Am J Physiol Renal Physiol, January 1, 2005; 288(1): F76 - F81. [Abstract] [Full Text] [PDF]

				S. Chen, J. A. McCormick, K. Prabaker, J. Wang, D. Pearce, and D. G. Gardner Sgk1 Mediates Osmotic Induction of NPR-A Gene in Rat Inner Medullary Collecting Duct Cells Hypertension, April 1, 2004; 43(4): 866 - 871. [Abstract] [Full Text] [PDF]

				S. C. Lenhard, S. S. Nerurkar, T. R. Schaeffer, R. C. Mirabile, R. W. Boyce, D. F. Adams, B. M. Jucker, and R. N. Willette p38 MAPK Inhibitors Ameliorate Target Organ Damage in Hypertension: Part 2. Improved Renal Function as Assessed by Dynamic Contrast-Enhanced Magnetic Resonance Imaging J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 939 - 946. [Abstract] [Full Text] [PDF]

				M. Kuhn Structure, Regulation, and Function of Mammalian Membrane Guanylyl Cyclase Receptors, With a Focus on Guanylyl Cyclase-A Circ. Res., October 17, 2003; 93(8): 700 - 709. [Abstract] [Full Text] [PDF]

				J. M. Capasso, C. J. Rivard, L. M. Enomoto, and T. Berl Chloride, not sodium, stimulates expression of the {gamma} subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells PNAS, May 27, 2003; 100(11): 6428 - 6433. [Abstract] [Full Text] [PDF]

				K. J. Cowan and K. B. Storey Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress J. Exp. Biol., April 1, 2003; 206(7): 1107 - 1115. [Abstract] [Full Text] [PDF]

				Q. Ye, S. Chen, and D. G. Gardner Endothelin Inhibits NPR-A and Stimulates eNOS Gene Expression in Rat IMCD Cells Hypertension, March 1, 2003; 41(3): 675 - 681. [Abstract] [Full Text] [PDF]

				R. Garg and K. N. Pandey Angiotensin II-Mediated Negative Regulation of Npr1 Promoter Activity and Gene Transcription Hypertension, March 1, 2003; 41(3): 730 - 736. [Abstract] [Full Text] [PDF]

				S. Chen, L. Cao, H. D. Intengan, M. Humphreys, and D. G. Gardner Osmoregulation of Endothelial Nitric-oxide Synthase Gene Expression in Inner Medullary Collecting Duct Cells. ROLE IN ACTIVATION OF THE TYPE A NATRIURETIC PEPTIDE RECEPTOR J. Biol. Chem., August 30, 2002; 277(36): 32498 - 32504. [Abstract] [Full Text] [PDF]

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