Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755
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ABSTRACT |
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The
Na+/H+ exchanger (NHE) regulates intracellular
pH, cell volume, Na+ absorption and H+
secretion in epithelial cells of the renal proximal tubule (PT). 1-Adrenergic receptors (ARs) increase NHE activity in PT
cells. The purpose of this study was to determine the mechanism of
1-AR activation of NHE isoforms expressed in PT cells.
Northern and Western blotting demonstrate transcripts and protein
expression of NHE1 and NHE3 in PT cells. An anti-NHE1 antibody
predominately labels protein expressed at basal and lateral membranes.
In contrast, NHE3 protein is expressed exclusively at the apical
membrane. To determine NHE isoforms regulated by
1-ARs,
antisense oligodeoxynucleotides (AS-ODNs) specific for NHE1 and NHE3
isoforms were introduced into cells with streptolysin O
permeabilization. Cells incubated with AS-ODNs a total of three
times exhibited a reduction in protein expression of ~85%. Na uptake
and changes in intracellular pH (pHi) were used as measures
of NHE activity in PT cells.
1-AR stimulation increased
Na uptake from 8.5 to 13.8 nmol · min
1 · mg protein
1.
AS-ODNs to NHE3 significantly reduced
1-AR stimulated Na
uptake and increases in pHi; no effect was observed in
sense-ODN-treated cells. Inhibition of NHE1 but not NHE3 expression
abolishes amiloride-suppressible NHE activity.
1-AR
stimulation of NHE1 is inhibited by the protein kinase C (PKC)
inhibitor calphostin C whereas NHE3 activity is abolished by the
mitogen-activated protein kinase (MAPK) inhibitor PD-98059. In PT cells
transfected with MAPK kinase MEKK1COOH, a truncated version
of MEKK1 that activates MAPK, NHE3 but not NHE1 activity is stimulated.
We conclude that
1-ARs activate distinct signaling
pathways to regulate specific NHE isoforms localized on opposite
membranes in polarized renal epithelial cells.
1-AR
activation of NHE1 is regulated by PKC whereas NHE3 is controlled by
MAPK and serves to separately regulate pHi, Na absorption,
and proton excretion in PT cells.
antisense oligonucleotide; intracellular pH; mitogen-activated protein kinase; sodium-hydrogen exchanger; protein kinase C; proximal tubule
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INTRODUCTION |
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IN RENAL EPITHELIAL
CELLS of the proximal tubule (PT) (15, 18),
1-adrenergic receptors (ARs) increase
Na+/H+ exchange
[Na+/H+ exchanger (NHE)]. NHE is specifically
regulated by
1A- and
1B-adrenergic receptor subtypes (28). The purpose of this investigation
was twofold: 1) to determine the NHE isoforms activated by
1-ARs in PT cells and 2) to identify the
signaling pathways generated by
1-ARs that activate
specific NHE isoforms.
In epithelial cells of the kidney, NHE plays an important role in regulation of intracellular pH (pHi), cell volume, Na+ absorption, and acid secretion (35). To date, six NHE isoforms derived from distinct genes have been characterized (32, 33). In epithelial cells, NHE1 is localized to basolateral membranes and serves a "housekeeping" function in regulation of pHi and cell volume (33, 35). NHE2 is localized to basolateral (38) and apical membranes (24) in distal nephron segments (10). NHE3 is the isoform localized to the apical membrane of PT and mediates Na+ and bicarbonate absorption and proton secretion (5, 26, 35). NHE4 is highly expressed in collecting duct and may regulate cell volume (7, 9). NHE1 is sensitive to inhibition by analogs of amiloride whereas the other isoforms are relatively resistant (7, 35).
We show that message and protein for NHE1 and NHE3 are expressed in
mouse PT cells. When visualized with confocal microscopy and specific
antibodies, NHE1 is localized to the basal and lateral membranes and
NHE3 to apical membranes of polarized PT cells. Stimulation of
1-ARs on these cells activates both NHE1 and NHE3 isoforms. To discern the signaling pathways utilized by
1-ARs to activate specific NHE isoforms, we used
complementary approaches that include antisense
oligodeoxynucleotides (AS-ODNs) to inhibit expression of each isoform,
pharmacological antagonists for specific signaling pathways, and
reconstitution of signaling pathways with transfection of
constitutively active mutants and pharmacological agents. Increases in
NHE activity were documented by using labeled Na uptake and changes in
pHi. We demonstrate that
1-AR activation of
NHE1 is protein kinase C (PKC) dependent whereas that for NHE3 is
mitogen-activated protein kinase (MAPK) dependent. We conclude that
activation of specific NHE isoforms by
1-ARs on PT cells is achieved through distinct signaling pathways. Basolateral NHE1 is
regulated by a PKC-dependent mechanism and participates in regulation
of intracellular pH. Apical membrane NHE3 is activated by a
PKC-independent MAPK pathway and facilitates net Na absorption and
proton secretion.
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METHODS |
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Preparation of primary cell cultures and immortalized cells. Primary cultures of mouse PT cells (proximal convoluted and straight tubules) and immortalized S1 PT cells were isolated and grown as previously reported (29, 36). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 media (Sigma, St. Louis, MO) supplemented with 5% fetal calf serum and antibiotics in a humidified atmosphere at 37°C (28). Similar conditions were used for culturing primary cultures of PT cells and S1 immortalized cells on plastic dishes for uptake studies, glass coverslips for measuring pHi, and for those grown on porous filters for use in confocal microscopy.
RT-PCR and sequencing.
Isolation of RNA from primary cultures and immortalized PT cells has
been described previously (28). Primers for NHE1, NHE2, NHE3, and NHE4 isoforms were designed and synthesized on the basis of
published rat cDNA sequences and are shown in Table
1 (34, 39). RT-PCR reactions
were performed by using total cellular RNA. As a control for genomic
DNA contamination of the RNA preparations, parallel samples were not
reverse transcribed. PCR products were electrophoresed on a 4%
low-melting agarose gel, stained with ethidium bromide, cut from the
gel, the cDNA was eluted, and each product was directly sequenced with
3.2 pmol of the forward or reverse PCR primers by using the PRISM
DyeDeoxy Sequencing Kit (Applied Biosytems, Foster City, CA)
(21).
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Northern blot analysis.
The radiolabeled cDNA probes from RT-PCR products were used to probe
RNA from mouse PT cells (21). The purified RT-PCR products were random-primer labeled by using [32P]dCTP (ICN, Costa
Mesa, CA). Total RNA from PT cells was electrophoresed on 1%
agarose/6% formaldehyde gels and then transferred to GeneScreen Plus
Hybridization Transfer Membrane overnight. After prehybridization with
2 × 106
counts · min1 · ml
1 of
radiolabeled probes and 100 µg/ml of salmon sperm DNA, the membrane
was incubated in hybridization solution for 20 h at 60°C.
Western blot analysis. Membrane protein from primary cultures of PT cells and immortalized S1 cells was prepared as previously described (28). Fractionated proteins were transferred to a nitrocellulose membrane and blocked with 5% Blotto in Tris-buffered saline (137 mM NaCl, 20 mM Tris, pH 7.4) at 4°C overnight. Blots were incubated with a monoclonal antibody (Ab) 4E9 for NHE1 (Dr. P. Aronson, Yale Univ.) (6) or a polyclonal Ab for NHE2 (Dr. M. Donowitz, Johns Hopkins Univ.) (24). A polyclonal Ab 1568 was used to identify NHE3 (Dr. O. W. Moe, Univ. of Texas at Dallas) (2), and a polyclonal Ab was used for NHE4 (amino acid sequence 609-618; Dr. R. Chambrey) (9). Each blot was incubated with Ab for 90 min at room temperature. Visualization was performed with 1:3,000 peroxidase-conjugated rabbit IgG fraction to mouse IgG (Cappel, Durham, NC) in Tris-buffered saline for 60 min and the enhanced chemiluminesence detection system (Amersham) and exposed on Kodak X-film.
Measurement of MAPK activity. Treatment of primary cultures of PT cells with phenylephrine (PHE) or inhibitors was performed by replacement of medium containing phosphatase inhibitors (orthovanadate, calyculin A) with or without drugs for 15 min. Cells were scraped and lysed into buffer containing protease inhibitors, then sonicated and centrifuged. An aliquot of supernatant was immunoprecipitated with anti-MAPK Ab (MAPK 1/2; Upstate Biotechnology) following the manufacturer's protocol. The complex was incubated with myelin basic protein substrate for 20 min at 30°C and run on a 7.5% SDS-PAGE gel, transferred to nitrocellulose, and incubated at 4°C overnight with mouse anti-phospho-myelin basic protein. Horseradish peroxidase conjugate incubation was followed by detection with enhanced chemiluminescence (Amersham).
Confocal microscopy to examine localization and expression of NHE1 and NHE3. Primary cultures of PT cells were grown on collagen I-coated cell culture inserts (1 µm pore size) and fixed with methanol-free paraformaldehyde in PBS solution. Cells were treated overnight with primary Ab at 4°C, rinsed three times, and incubated with secondary Ab coupled to Alexa 488 or Alexa 568 (Molecular Probes). The filter was mounted on a glass coverslip with ProLong Antifade reagent (Molecular Probes). The slide was visualized by using a Zeiss microscope attached to a Bio-Rad model MRC-1024 scanning confocal system equipped with three photomultiplier tube detectors with Kr/Ar laser excitation at 488, 568, and 647 nm. Confocal images were digitally acquired into two-dimensional arrays and assigned intensities of 0-255. Three-dimensional reconstruction was performed by using Bio-Rad imaging or National Institutes of Health Image software.
Design and introduction of AS-ODNs. ODNs were designed from the cDNA sequence obtained from mouse PT cells used in this study. Antisense and sense 18-mers were phosphorothioate substituted in each position. The sequences for the oligos are 5'-CGATGAGGCAGAAGAGCA-3' (NHE1 antisense; positions 80-97); 5'-TGCTCTTCTGCCTCATCG-3' (NHE1 sense); 5'-ACTCGCACAGAACCCACA-3' (NHE2 antisense; positions 56-73); 5'-TGTGGGTTCTGTGCGAGT-3' (NHE2 sense); 5'-CTGCTTTTCATCCTCATT-3' (NHE3 antisense; positions 98-115); 5'-AATGAGGATGAAAAGCAG-3' (NHE3 sense); 5'-ACTGATAGGGTGTGGGAG-3' (NHE4 antisense ;positions 143-160); and 5'-CTCCCACACCCTATCAGT-3' (NHE4 sense). ODNs were introduced into cells with transient streptolysin O permeabilization (4). Control cells were permeabilized with streptolysin O but did not receive ODNs. The time course and concentration dependence for inhibition of protein expression were determined for each isoform with Western blotting. Maximal inhibition of protein expression was achieved by using three treatments with 5 µM antisense ODNs over 72 h.
Transfection with MAPK kinase MEKK1COOH.
Primary cultures of PT cells were transfected with the active mutant
MEKK1COOH, a truncated version of MEKK1 (22)
generously provided by G. L. Johnson (27). PT cells
in culture were cotransfected with MEKK1COOH plasmid (7 µg plasmid/60-mm dish) and -galactosidase (7 µg plasmid/60-mm
dish; Promega) or
-galactosidase alone by using a calcium phosphate
technique (11). Cell viability was assessed by trypan blue
exclusion, and transfection efficiency, based on
-galactosidase
staining, was estimated to be ~70%.
22Na+ uptake as a measure of NHE activity. A rapid filtration technique described in previous reports (18, 19) was used to monitor NHE exchange activity in primary cultures of PT cells or immortalized S1 cells. The uptake of 22Na+ into PT cells at 37°C that is inhibited by the specific blocker ethylisopropyl amiloride (EIPA; 10 µM), is the amount of Na that enters cells through NHE. Control experiments indicate that tracer uptake is linear for 2 min. Tracer uptake was terminated after 1 min by rapid addition of ice-cold isoosmotic buffer and filtered onto Whatman GF/C filters by using a Millipore 12-port manifold. Nonspecific binding of 22Na+ to filters and cells was determined and subtracted to calculate uptake.
Determination of pHi. Primary cultures of PT cells were grown on 25-mm glass coverslips and incubated with the pH-sensitive dye BCECF-AM as previously described (28). Cells on coverslips were placed in a temperature-controlled chamber of microincubation system at 37°C (16). A Nikon Photoscan-2 (Nikon, Natick, MA) was used to measure fluorescence intensity. Each experiment was calculated by using equilibration with buffers of varying pH values (6.5-7.6) and valinomycin.
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RESULTS |
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Analysis of NHE isoform transcripts in PT cells. RT-PCR for each primer set was performed on RNA samples obtained from four independent isolations. Primers for NHE1 produced a band of 314 bp, 222 bp for NHE2, 296 bp for NHE3, and 437 bp for NHE4. For all primer sets and RNAs used, samples analyzed in the absence of RT resulted in no product. We report that mouse PT cells express transcripts encoding all four NHE isoforms. To confirm the identity of PT NHE isoforms, PCR products were sequenced. Partial clones from PT cells demonstrated high similarity to published sequences (12, 34, 39). The mouse NHE1 product was 100% identical at the amino acid level (96% nucleotide identity) with published rat sequences (34). Amino acid sequences for NHE2, NHE3, and NHE4 products were 95, 97, and 96% identical to reported sequences (12, 34, 39), respectively.
Northern blot analysis was used to determine sizes of NHE isoform mRNAs in PT cells. Figure 1A shows a transcript of 4.4 kb obtained with the NHE1 probe and hybridization of mRNA from primary cultures of PT cells and immortalized S1 cells. This size is identical to that in kidney cortex, PT, and LLC-PK1 cells (3). A transcript was detected for NHE3 in PT and S1 cells and was similar in size to that observed in opossum kidney cells (31). Larger transcripts were observed with mRNA of kidney and other tissues (3, 34). A transcript of 3.9 kb was observed with the NHE2 probe, and a band of 4.2 kb was observed with the NHE4 probe and mRNA from PT and S1 cells (data not shown).
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Determination of NHE isoform protein expression. Two complementary methods were used to examine protein expression of NHE isoforms on PT: 1) Western blot analysis with membrane preparations from freshly isolated, primary cultures and immortalized S1 cells; and 2) immunofluorescence. Results of Western blotting with membranes prepared (day 0, no AS-ODN treatment) from primary cultures of PT cells is shown in Fig. 1B. A monoclonal antibody anti-fpNHE1-514/818 (clone 4E9) identifies PT NHE1 protein (37). A 102-kDa protein was detected with membrane from primary cultures of PT cells; a similar band was observed with membrane protein isolated from immortalized S1 cells. This band corresponds to the mature form of this isoform reported in LAP1 cells (37). A polyclonal anti-peptide Ab generated to rat NHE3 was used to examine protein expression in mouse PT cells (2). This antibody specifically recognizes NHE3. A doublet at 85 and 87 kDa was detected with membrane from primary cultures of PT cells (Fig. 1B; time 0, no AS-ODN treatment) and also observed in immortalized S1 cells. This observation is consistent with labeling of 87-kDa proteins in membrane of rat kidney cortex and medulla (2).
Protein expression for NHE isoforms was also examined by immunofluorescent labeling. As shown in Fig. 2, distinct areas of punctate labeling were observed by using confocal microscopy of NHE1 and NHE3 to detect labeling in primary cultures of PT cells (and in immortalized S1 cells). Negligible labeling was observed with secondary antibody alone. Immunofluorescent labeling of NHE1 and NHE3 was also evident in primary cultures of PT cells grown on glass coverslips (data not shown). Very weak immunofluorescence was detected for NHE2 and NHE4 labeling in PT cells. We conclude that transcripts for NHE2 and NHE4 are present in rat PT cells, but protein expression for these two isoforms is very low. This is consistent with the low levels of membrane expression for NHE2 and NHE4 isoforms detected in rat PT cells (9, 10).
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NHE isoforms activated by 1-adrenergic receptors.
We previously reported that
1A- and
1B-AR
subtypes activate NHE in PT cells (28). To assess the NHE
isoforms regulated by
1-ARs, we treated cells with
antisense specific for NHE1 and NHE3 isoforms. Cells were treated with
5 µM concentrations of AS-ODNs three times over 72 h because
this treatment produces a maximal reduction in NHE protein expression.
Data presented in Fig. 1 show that protein expression of NHE1 and NHE3
is progressively reduced relative to
-actin with AS-ODN treatment
that is specific for each isoform. No reduction in protein expression
was observed with sense NHE1 and NHE3 treatments over the course of 3 days. Similarly, when the blots were probed with the antibody specific for the other isoform, there was no significant reduction in protein expression. Cells treated with NHE1 AS-ODNs did not exhibit reduced levels of NHE3 protein, nor did cells treated with NHE3 AS-ODNs show
any evidence of reduced expression of NHE1. The inhibition of protein
expression was 86 ± 9 and 84 ± 13% (n = 5 separate experiments; Fig. 1C) for NHE1 and NHE3 isoforms,
respectively, with 3 days of AS-ODN treatment.
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PKC and MAPK selectively regulate NHE isoforms.
To understand the differential regulation of NHE1 and NHE3 isoforms by
1-ARs in PT cells, we examined signaling pathways activated by
1-ARs. The primary signaling pathways
utilized by
1-ARs are phosphatidylinositol-specific
phospholipase C (PI-PLC) and stimulation of PKC, MAPK, and calcineurin
(13, 23, 42). To test this in PT cells, we measured the
effects of activation and inhibition on NHE isoforms. In Fig.
6 we show that the PKC inhibitor
calphostin C reduces PHE-stimulated Na uptake by ~25% but reduces
the basal rate of Na uptake by ~50%. By comparison, the component of
PHE-stimulated Na entry is nearly equivalent for untreated cells and
those treated with calphostin C. These findings suggest that the basal
rate of Na entry through NHE is inhibited by calphostin C but that the
stimulated portion remains unchanged. In cells pretreated with the MAPK
inhibitor PD-98059, PHE-stimulated uptake is nearly abolished. With
combined treatment of calphostin C and PD-98059, PHE-stimulated and
basal rates of Na uptake are significantly inhibited. These data
provide compelling evidence that stimulation of PKC is required for
activation of NHE1, and MAPK is required for activation of NHE3.
PD-98059 has no effect on the basal rate of uptake whereas calphostin C
has no effect on the PHE-stimulated rate of uptake. The results are not
as clear cut with PHE-induced changes in pHi (Fig.
7). Under these conditions, changes in
pHi represent net changes due to combined NHE1 and NHE3
present in the membrane. The PHE-stimulated increase in pHi
is significantly reduced with PD-98059 treatment, but there is also
inhibition with calphostin C. The reduction in PHE-stimulated
pHi that occurs with calphostin C treatment may result from
the inability of the cell to properly regulate pHi through
NHE1. To test if calcineurin, a Ca-dependent protein phosphatase 2B, is
involved in
1-AR regulation of NHE in PT cells (23), we used the calcineurin inhibitors FK-506 and
cyclosporine. No significant changes in basal,
1-AR-stimulated, or EIPA-inhibited rates of Na entry
were observed with these inhibitors.
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DISCUSSION |
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The purpose of the present study was 1) to distinguish
NHE isoforms present in PT cells; 2) to determine NHE
isoforms activated by 1-ARs in these cells; and
3) to identify signaling pathways that stimulate NHE
isoforms. NHE1 message is expressed in virtually all tissues
(41). NHE3 transcripts are expressed in epithelial tissues, with high levels in kidney and intestine (8).
Message for NHE2 and NHE4 is also expressed in epithelial cells of the kidney (7, 10). We identified transcripts for each of the four NHE isoforms in mouse PT cells but only observed significant expression of NHE1 and NHE3 protein (Fig. 1). This is in agreement with
findings in rats that show NHE2 protein is expressed at very low levels
in proximal nephron compared with distal nephron (10). We
detected NHE2 protein expression in distal segments (data not shown)
and very low levels of NHE2 in PT cells. Our observations that NHE4
protein is expressed at very low levels in PT cells is consistent with
lack of expression of NHE4 protein in rat PT and other renal epithelial
cells (9, 44).
Labeling with isoform-specific antibodies and visualization with
confocal microscopy demonstrated that NHE1 is expressed on basolateral
membranes whereas NHE3 is expressed on apical membranes of PT. Our
findings are consistent with other reports (2, 6). The
distinct targeting of these proteins in polarized epithelial cells
suggests that NHE isoforms serve specific functions. We propose that
1-ARs utilize specific signaling pathways to stimulate NHE1 and NHE3 isoforms on spatially separated, polarized membranes of
PT cells. Given the unique distribution of NHE isoforms in renal
epithelia, we sought to determine the isoforms activated by
1-ARs in PT cells. To discern specific NHE isoforms
activated by
1-ARs in PT cells, AS-ODNs were used to
inhibit protein expression for each isoform. Using labeled Na uptake
and changes in pHi as two independent methods to assess
NHE, we demonstrated that
1-ARs activate NHE1 and NHE3
isoforms in PT cells. Inhibition of protein expression for NHE isoforms
is specific to the AS-ODN used to treat the cells (Fig. 1B).
An average of 85% reduction in NHE1 and NHE3 protein expression was
observed in cells treated three times with AS-ODNs. AS-ODNs to NHE1 and
NHE3 significantly inhibited PHE-stimulated Na uptake and increases in
pHi (Figs. 3-5; Table 3). We observed no reduction in
PHE-stimulated uptake with AS-ODN reduction in NHE2 and NHE4 protein
expression. This is consistent with low levels of protein expression
observed in PT cells.
EIPA-sensitive Na uptake was abolished only with AS-ODNs to NHE1 (Figs. 3 and 4). Several reports show that NHE2-4 isoforms are much less sensitive or resistant to inhibition by amiloride analogs (30). Our findings that NHE3 AS-ODN treatment does not inhibit EIPA-sensitive uptake is consistent with resistance of this isoform to inhibition by amiloride analogs.
The ubiquitous NHE1 isoform is present on basolateral membrane of PT cells (14). NHE1 is designated as the housekeeping exchanger and is postulated to regulate pHi (41). The basolateral distribution in PT cells suggests another isoform mediates apical Na absorption. NHE3 is localized to the apical membrane of these cells (2). The distinct localization and activation of these transporters by receptor signaling pathways suggest that the two isoforms perform different functions.
1-ARs in PT cells couple to several second messengers.
These receptors couple through G
q/11 proteins
and PI-PLC that in turn activate PKC (28). Recent data
show that
1-ARs also activate MAPK (43) and
calcineurin (23). Short-term activation of PKC acutely
increases NHE1 activity whereas long-term activation results in a
chronic and persistent increase in NHE activity (1).
Paradoxically, phorbol esters acutely increase NHE3 activity in PT
epithelial cells but inhibit activity in other cell types
(1). This may be attributable, in part, to the unique
environment of the PT cell whereby regulation of pHi and
transepithelial movement of Na occur simultaneously. NHE activity is
stimulated by some hormones (
-ARs, angiotensin II) and inhibited by
others (parathyroid hormone, dopamine) (1). Stimulation of
1-ARs on PT cells by renal nerves or circulating
catecholamines permits acute activation of NHE to facilitate movement
of Na and protons and regulation of pHi.
We propose that 1-ARs activate specific isoforms through
generation of distinct second messengers. As depicted in Fig. 6, inhibitors of PKC and MAPK are required to abolish
1-AR-induced NHE activity. We observed no effect on
basal, PHE-stimulated, or EIPA-inhibited rates of Na entry in cells
treated with selective antagonists of calcineurin. The MAPK inhibitor
PD-98059 eliminated a majority of
1-AR-induced Na entry.
By comparison, calphostin C inhibited the bulk of the
1-AR-induced increase in pHi (Fig. 7). These
observations suggest that apical Na entry is mediated by NHE3 and
activated by MAPK whereas basolateral NHE1 participates in the
regulation of pHi stimulated by PKC. In PT cells
transfected with MEKK1COOH, a truncated version of MEKK1
that strongly activates MAPK (22), NHE activity was
significantly increased (Fig. 8). The increased rate of Na uptake
observed with MEKK1COOH transfection is similar to the rate
observed with the
1-AR agonist PHE. In primary cultures
of PT cells treated with NHE3 AS-ODNs, the increased rate of Na entry
due to activation of MAPK with MEKK1COOH transfection was
abolished. By comparison, Na entry in cells transfected with MEKK1COOH and treated with NHE1 AS-ODNs is equivalent to
that observed in control cells that do not receive AS-ODNs. We
attribute the decreased Na entry rate with NHE3 AS-ODN treatment to a
reduction in NHE3 protein expression and activity. These findings
provide compelling evidence that
1-AR activation of MAPK
almost exclusively stimulates NHE3 but has little effect on NHE1. In
complementary experiments, we used phorbol ester to activate PKC. The
results show that treatment with NHE1 AS-ODNs abolishes PMA-stimulated uptake and that this component is equivalent to the EIPA-sensitive component of Na uptake (Fig. 8). Rates for EIPA-inhibited Na entry were
similar in cells treated with NHE3 AS-ODNs compared with the rate in
control cells that did not receive AS-ODNs.
We postulate that 1-AR activation of signaling pathways
for PKC and MAPK occurs independently in PT cells. As depicted in Fig.
9,
1-AR stimulation of PI-PLC and PKC is not required
for increases of MAPK activity. The PI-PLC inhibitor U-73122 and PKC inhibitor calphostin C had no effect on PHE-induced MAPK activity whereas activity was abolished with PD-98059. Activation of MAPK may
occur through a Ras-dependent pathway that does not involve PKC but
through other kinase phosphorylate Shc adapter proteins and coordinate
Grb2/SOS proteins with the Ras complex (25). Our results
are consistent with other reports that show activation of PKC is not
required for activation of MAPK in PT cells. In these experiments we
used an anti-phospho MAPK kinase (Erk 1/2) antibody that is specific
for dually phosphorylated Erk 1 (p44) and Erk 2 (p42). In Fig.
9B we depict the summary for activation of p44 and p42. It
appears that both MAPK forms are activated on stimulation of
1-ARs in PT cells. We conclude that
1-ARs independently stimulate PI-PLC and PKC and MAPK pathways to selectively enhance NHE1 and NHE3 activity in PT cells.
In summary, we show that two NHE isoforms predominate in PT epithelial
cells: NHE1 and NHE3. Activation of 1-ARs specifically activates NHE1 and NHE3 isoforms. Given the unique apical and basolateral distribution of transporters in these cells, we speculate that
1-ARs increase absorption of apical Na and
secretion of protons through NHE3 and regulate pHi by NHE1.
To achieve this goal,
1-ARs employ distinct signaling
pathways to activate NHE1 and NHE3. Increases in NHE1 activity occur
through PI-PLC and PKC second messengers whereas NHE3 activity is
increased through stimulation of MAPK pathways independently of PKC in
PT cells.
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ACKNOWLEDGEMENTS |
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The authors thank Bonita A. Coutermarsh for expert technical assistance. The authors also thank Dr. Peter Aronson (Yale University), Dr. Mark Donowitz (Johns Hopkins University), Dr. Regine Chambrey (Institut National de la Santé et de la Recherche Médicale 356, Université Pierre et Marie Curie, Paris, France), and Dr. Orson Moe (University of Texas at Dallas) for sharing isoform-specific antibodies. Gary L. Johnson (University of Colorado Medical School) is acknowledged for his generous contribution of the MEKK1COOH plasmid.
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FOOTNOTES |
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This work was supported in part by an American Society of Nephrology Career Enhancement Award and National Institute of Diabetes and Digestive and Kidney Diseases Training Grant DK-07301.
Address for reprint requests and other correspondence: F. A. Gesek, Dept. of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755 (E-mail: fg{at}Dartmouth.edu).
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.
Received 17 April 2000; accepted in final form 1 November 2000.
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