alpha 1-Adrenergic receptors activate NHE1 and NHE3 through distinct signaling pathways in epithelial cells

Fengming Liu and Frank A. Gesek

Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Na+/H+ exchanger (NHE) regulates intracellular pH, cell volume, Na+ absorption and H+ secretion in epithelial cells of the renal proximal tubule (PT). alpha 1-Adrenergic receptors (ARs) increase NHE activity in PT cells. The purpose of this study was to determine the mechanism of alpha 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 alpha 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. alpha 1-AR stimulation increased Na uptake from 8.5 to 13.8 nmol · min-1 · mg protein-1. AS-ODNs to NHE3 significantly reduced alpha 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. alpha 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 alpha 1-ARs activate distinct signaling pathways to regulate specific NHE isoforms localized on opposite membranes in polarized renal epithelial cells. alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN RENAL EPITHELIAL CELLS of the proximal tubule (PT) (15, 18), alpha 1-adrenergic receptors (ARs) increase Na+/H+ exchange [Na+/H+ exchanger (NHE)]. NHE is specifically regulated by alpha 1A- and alpha 1B-adrenergic receptor subtypes (28). The purpose of this investigation was twofold: 1) to determine the NHE isoforms activated by alpha 1-ARs in PT cells and 2) to identify the signaling pathways generated by alpha 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 alpha 1-ARs on these cells activates both NHE1 and NHE3 isoforms. To discern the signaling pathways utilized by alpha 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 alpha 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 alpha 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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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Table 1.   Primers used for amplification of NHE isoform transcripts by RT-PCR

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 · min-1 · 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 beta -galactosidase (7 µg plasmid/60-mm dish; Promega) or beta -galactosidase alone by using a calcium phosphate technique (11). Cell viability was assessed by trypan blue exclusion, and transfection efficiency, based on beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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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Fig. 1.   Northern and Western blots of mouse proximal tubule (PT) cell RNA and membrane protein for Na/H+ exchanger (NHE) isoforms. A: total RNA from primary cultures of PT or S1 cells probed with [32P]dCTP-labeled PCR products for NHE1 and NHE3 isoforms. B: NHE protein expression determined by using Western blotting with membrane protein obtained from primary cultures of mouse PT using a monoclonal antibody (Ab) 4E9 for NHE1 (courtesy of Dr. P. Aronson) and polyclonal Ab 1568 generated to rat NHE3 (courtesy of Dr. O.W. Moe). Protein was obtained from primary cultures of untreated PT cells (day 0) and those treated for 1-3 days with antisense (AS)- or sense oligodeoxynucleotides (ODNs). Blots were then probed with NHE1, NHE3, or beta -actin-specific antibodies. C: summary of the reduction in protein expression for NHE1 and NHE3 with 1, 2, or 3 treatments with AS-ODNs. Bars denote means ± SE for 3-4 separate treatments for each condition determined from densitometry relative to beta -actin.

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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Fig. 2.   Localization of NHE1 and NHE3 isoforms in PT cells. Primary cultures of PT cells were grown on filter inserts and incubated with a monoclonal Ab 4E9 for NHE1 and polyclonal Ab 1568 for NHE3; secondary antibodies coupled to Alexa 488 or Alexa 568 were used to visualize labeling, respectively. Top: NHE3 protein viewed primarily along the apical membrane of PT cells. Bottom: expression of NHE1 along basal and lateral membranes of primary cultures of PT cells.

NHE isoforms activated by alpha 1-adrenergic receptors. We previously reported that alpha 1A- and alpha 1B-AR subtypes activate NHE in PT cells (28). To assess the NHE isoforms regulated by alpha 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 beta -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.

EIPA selectively inhibits the NHE component of Na+ uptake in renal tubule cells (17, 20, 40). As depicted in Fig. 3, the EIPA-suppressible component represents ~60% of the basal rate of 22Na+ influx and compares favorably with previous studies (20). Comparable treatment of PT cells with 10 µM amiloride attenuated the basal rate of Na uptake by ~50%. A concentration of 100 µM amiloride inhibited the basal rate of Na entry by 68%, which was not significantly different from the inhibition observed with 10 µM EIPA. Treatment of cells with NHE1 AS-ODNs significantly inhibited the EIPA sensitivity of Na entry. At 3 days there was virtually no EIPA-suppressible component of Na+ entry. The basal rate of NHE activity was reduced with increased treatment of NHE1 AS-ODNs from 1 to 3 days and reflects the loss of NHE1 activity. These findings provide evidence that the EIPA-suppressible component of NHE is largely composed of NHE1 and, under basal conditions, the bulk of NHE activity in these cells is mediated by NHE1. Cells treated for 3 days with AS-ODNs to NHE3 exhibited no reduction in control or EIPA-suppressible Na uptake. These findings are consistent for NHE3 being an "amiloride-resistant" isoform that is much less sensitive to EIPA than NHE1 or NHE2.


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Fig. 3.   Na uptake stimulated by alpha 1-adrenergic receptors (ARs) is inhibited in cells treated with NHE3 antisense treatment whereas ethylisopropyl amiloride (EIPA)-sensitive entry is inhibited by NHE1 antisense. The percent change is calculated from the basal rate obtained on each treatment day. A: phenylephrine (PHE)-stimulated (1 µM) rates. , Primary cultures of PT cells treated with NHE1 AS; , Primary cultures of PT cells treated with NHE3 AS. B: EIPA (10 µM)-sensitive portion of Na uptake in PT cells treated with NHE1 and NHE3 AS-ODNs. Symbols denote mean ± SE for 4 separate experiments with triplicate measurements for each point. *P < 0.01 compared with PHE-stimulated and EIPA-inhibited rates from untreated cells.

To determine the NHE isoform regulated by alpha 1-ARs, the alpha 1-selective agonist PHE was used to activate the receptor. PHE increased Na uptake an average of 73% relative to the control rate of uptake. As depicted in Fig. 3, 2- and 3-day treatments with NHE3 AS-ODNs significantly reduced PHE-stimulated Na uptake. No inhibition of PHE-induced uptake was observed with NHE1 AS-ODN treatments. For both NHE1 and NHE3, rates of uptake in sense ODN-treated cells were not significantly different from control (for untreated cells, the rate is 8.9 ± 0.3 nmol · min-1 · mg protein-1 compared with three treatments of NHE1 or NHE3 sense ODNs: 9.1 ± 0.2 and 8.7 ± 0.3 nmol · min-1 · mg protein-1, respectively). There is no significant difference in PHE-stimulated rates among control cells or those treated with NHE1 or NHE3 sense ODNs for 3 days (14.1 ± 0.3, 13.8 ± 0.3, 14.3 ± 0.2 nmol · min-1 · g protein-1 for control, NHE1 sense ODN, and NHE3 sense ODN treatments, respectively). Figure 4 summarizes inhibition with NHE1, NHE3, and combined NHE1+NHE3 AS-ODNs on PHE-stimulated and EIPA-inhibited components of Na uptake. Treatment with NHE1 AS-ODNs clearly reduces EIPA-inhibitable uptake whereas NHE3 AS ODN treatment significantly inhibits PHE-stimulated uptake. With combined NHE1+NHE3 AS-ODN treatments, both PHE-stimulated and the EIPA-inhibitable components of entry are abolished. The rate of PHE-stimulated or EIPA-inhibited Na uptake is not significantly different in cells treated with NHE2 or NHE4 AS-ODNs compared with control cells (data not shown).


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Fig. 4.   NHE3 AS-ODN treatment blocks alpha 1-AR-stimulated Na entry whereas NHE1 AS-ODN treatment blocks EIPA-suppressible Na uptake in primary cultures of PT cells. Percent change is calculated from the basal rate from primary cultures of PT cells receiving no oligo treatment. For each condition, cells were treated 3 times over 72 h with NHE1, NHE3, or combined NHE1+NHE3 AS-ODNs. Bars denote mean ± SE for 4 separate experiments with triplicate measurements for each point. *P < 0.01 compared with EIPA-inhibited and PHE-induced changes in cells receiving no oligo treatments.

As depicted in Table 2, treatment of cells for 3 days with NHE1 and NHE3 AS-ODNs significantly reduces PHE-stimulated rates of change and steady-state pHi compared with responses of control cells. Although there is a significant reduction in the PHE-stimulated rates with NHE1 and NHE3 AS-ODN-treated cells, steady-state changes in pHi are not fully inhibited to basal levels. The incomplete block of PHE-stimulated pHi changes may result from compensation due to the presence of the NHE1 isoform. With combined NHE1 and NHE3 AS-ODN treatment, the PHE-stimulated rate of pHi change is very small, and the steady-state pH appears equivalent to that of basal pHi. As depicted in Fig. 5, when cells are treated with combined NHE1 and NHE3 AS, both PHE-stimulated rate of increase and steady-state increases of pHi are abolished.

                              
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Table 2.   Inhibition of NHE1 and NHE3 protein expression reduces alpha 1-AR-induced increases in intracellular pH



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Fig. 5.   alpha 1-AR-induced increases of intracellular pH are reduced in primary cultures of PT cells treated with NHE1 and NHE3 AS-ODNs. Tracings represent changes in intracellular pH in PT cells measured with the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM). After basal measurement, PHE (1 µM) was added to the incubation chamber. PT cells were treated with NHE1, NHE3, or combined NHE1+NHE3 AS-ODNs 3 times over 72 h before being loaded with BCECF and determination of intracellular pH.

Cells treated with NHE1 AS-ODNs exhibit a significant reduction in EIPA-sensitive Na uptake. As depicted in Fig. 3, the inhibition observed with EIPA in untreated cells is abolished with NHE1 AS-ODN treatment. Inhibition of NHE3 expression has no effect on the EIPA-suppressible component of uptake. NHE1 is sensitive to amiloride analogs whereas NHE2 is perhaps 10 times less sensitive and NHE3 and NHE4 appear to be resistant to amiloride analogs (39, 41). AS-ODNs to NHE2 and NHE4 isoforms exhibit no reduction in EIPA-suppressible or PHE-stimulated Na uptake (data not shown). The lack of functional effects is consistent with the low level of protein expression for NHE2 and NHE4 isoforms in PT cells.

PKC and MAPK selectively regulate NHE isoforms. To understand the differential regulation of NHE1 and NHE3 isoforms by alpha 1-ARs in PT cells, we examined signaling pathways activated by alpha 1-ARs. The primary signaling pathways utilized by alpha 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 alpha 1-AR regulation of NHE in PT cells (23), we used the calcineurin inhibitors FK-506 and cyclosporine. No significant changes in basal, alpha 1-AR-stimulated, or EIPA-inhibited rates of Na entry were observed with these inhibitors.


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Fig. 6.   Inhibition of mitogen-activated protein kinase (MAPK) blocks alpha 1-AR-induced NHE activity in PT cells. NHE activity was determined by using 22Na uptake in cells pretreated for 15 min with the protein kinase C (PKC) inhibitor calphostin C (100 nM) or the MAPK inhibitor PD-98059 (10 µM). NHE activity was stimulated in the presence and absence of inhibitors with PHE (1 µM). Bars denote means ± SE for 4 separate experiments with triplicate determinations of each maneuver. *P < 0.01 compared with PHE stimulation in cells receiving no oligos.



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Fig. 7.   Changes in intracellular pH are inhibited in PT cells treated with PKC and MAPK inhibitors. Increases of intracellular pH in response to PHE (1 µM) are reduced in PT cells treated with the MAPK inhibitor PD-98059 (10 µM) or the PKC inhibitor calphostin C (100 nM). Tracings were recorded in cells loaded with the pH-sensitive dye BCECF-AM (10 µM) for 1 h.

To determine the specific signaling pathways that activate NHE1 and NHE3 isoforms in PT cells, we selectively activated PKC and MAPK with low concentrations of phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or transfection of the active mutant MEKK1COOH, a truncated version of MEKK1 (22). In Fig. 8, we demonstrate that primary cultures of PT cells treated with PMA or transfected with MEKK1COOH increase Na uptake comparably to those stimulated by alpha 1-ARs. To discern the specific signaling pathways that activate NHE1 and NHE3 isoforms, we treated PT cells with NHE1 and NHE3 AS-ODNs to selectively inhibit expression of each isoform. A nearly complete inhibition of MEKK1-stimulated Na uptake was observed in primary cultures of PT cells treated with NHE3 AS-ODNs. The level of inhibition is comparable to that observed with alpha 1-AR-agonist-stimulated uptake in cells treated with NHE3 AS-ODNs (Fig. 4) and PD-98059 (Fig. 6). In contrast, PT cells treated with 100 nM PMA plus NHE1 AS-ODNs exhibited a significant reduction in Na uptake and approached the rate observed with NHE1 AS-ODN treatment alone. Interestingly, PMA did not significantly increase Na entry in S1 immortalized cells compared with primary cultures of PT cells. The basal rate of Na entry was inhibited by both NHE1 AS and calphostin C in S1 immortalized cells (data not shown). This is one apparent difference between the freshly isolated and primary cultures of PT cells from mice compared with the immortalized mouse S1 cell line. By comparison, the MEKK1-stimulated component of entry in NHE1 AS-ODN-treated primary cultures of PT cells is approximately equivalent to that observed in cells not treated with ODNs. In cells treated with combined NHE1 and NHE3 AS-ODNs, activation by either MEKK1 or PMA of MAPK and PKC pathways, respectively, is inhibited (data not shown). These findings provide additional proof that pathways involving MAPK in PT cells stimulate NHE3 and activation of PKC regulates NHE1.


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Fig. 8.   MAPK activates NHE3 but not NHE1 isoforms in primary cultures of PT cells. Stimulation of NHE in cells occurs in primary cultures of PT cells treated with phorbol ester [phorbol 12-myristate 13-acetate (PMA; 100 nM)] or transfected with the constitutively active mutant MEKK1COOH. PMA-stimulated Na entry is abolished in cells treated with NHE1 AS-ODNs but not NHE3 AS-ODNs. By comparison, cells transfected with active MEKK1 exhibit significant reductions in stimulated Na uptake with NHE3 AS-ODN treatment but are not affected by NHE1 AS treatment. Bars represent mean ± SE for n = 4 separate experiments with triplicate measurements for each treatment. P < 0.01 compared with PMA treatment or MEKK1 transfection in cells receiving no oligos.

Figure 9 shows that activation of alpha 1-ARs by PHE increases MAPK phosphorylation in PT cells. The MAPK inhibitor PD-98059, but not the PLC antagonist U-73122, blocked activation of MAPK by PHE. This suggests activation of PI-PLC is not required for activation of MAPK. Furthermore, pretreatment of cells with the PKC inhibitor calphostin C abolished PHE-induced increases in MAPK activity. This finding demonstrates that neither PI-PLC nor PCK is required for alpha 1-AR induced increases of MAPK in PT cells. The fact that MAPK does not require PKC activation affords a mechanism whereby stimulation of alpha 1-ARs can selectively activate NHE1 or NHE3 mechanisms.


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Fig. 9.   MAPK inhibitors inhibit alpha 1-AR-induced MAPK phosphorylation in primary cultures of PT cells. A: membranes from PT cells obtained after treatment with PHE (10 µM) alone or in combination with the MAPK inhibitor PD-98059 (10 µM), the PKC inhibitor calphostin C (100 nM), or the phospholipase C (PLC) inhibitor U-73122 (10 µM). A: immunoprecipitated MAPK obtained from treated and untreated cells blotted with phospho-specific MAPK1/MAPK2 antibody. Bands are observed at p42 and p44, respectively. B: summary of the results of 3 separate experiments for p44 and p42 activity measured relative to beta -actin. Bars denote means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was 1) to distinguish NHE isoforms present in PT cells; 2) to determine NHE isoforms activated by alpha 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 alpha 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 alpha 1-ARs in PT cells. To discern specific NHE isoforms activated by alpha 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 alpha 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.

alpha 1-ARs in PT cells couple to several second messengers. These receptors couple through Galpha q/11 proteins and PI-PLC that in turn activate PKC (28). Recent data show that alpha 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 (alpha -ARs, angiotensin II) and inhibited by others (parathyroid hormone, dopamine) (1). Stimulation of alpha 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 alpha 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 alpha 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 alpha 1-AR-induced Na entry. By comparison, calphostin C inhibited the bulk of the alpha 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 alpha 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 alpha 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 alpha 1-AR activation of signaling pathways for PKC and MAPK occurs independently in PT cells. As depicted in Fig. 9, alpha 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 alpha 1-ARs in PT cells. We conclude that alpha 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 alpha 1-ARs specifically activates NHE1 and NHE3 isoforms. Given the unique apical and basolateral distribution of transporters in these cells, we speculate that alpha 1-ARs increase absorption of apical Na and secretion of protons through NHE3 and regulate pHi by NHE1. To achieve this goal, alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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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