Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe

Rebecca L. McSwine1, Mark W. Musch1, Crescence Bookstein1, Yue Xie1, Mrinalini Rao2, and Eugene B. Chang1

1 Section of Gastroenterology, Department of Medicine, University of Chicago, Chicago 60637; and 2 Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

We examined the regulation of the Na+/H+ exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane 22Na+ influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-(N,N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.

Caco-2; electroneutral sodium absorption; brush-border membrane; transport proteins; nutrient transport; second messengers; signal transduction; intestinal transport; electrolyte transport; intracellular pH

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

APICAL MEMBRANE Na+/H+ exchange of intestinal epithelial cells is a major route for electroneutral, non-nutrient-dependent Na+ absorption. In contrast to the ubiquitously expressed basolateral membrane Na+/H+ exchanger (NHE) 1, brush-border membrane NHE is active even under basal conditions in the intestine, a teleologically important difference as this transport pathway must be operational whenever luminal Na+ is present. Apical membrane NHE activity can also be regulated by a number of signal transduction pathways. For instance, activation of the protein kinase C (PKC) pathway and increases in intracellular Ca2+, cGMP, and cAMP result in an inhibition of intestinal apical membrane NHE activity, with subsequent decreases in vectorial Na+ absorption (1, 8, 14, 17, 29).

In the past several years, two NHE isoforms have been identified, NHE2 and NHE3, which are predominantly expressed by epithelial cells, apically localized, and found throughout the intestine of various species (4, 7, 15, 19). Until recently, their relative contributions to overall vectorial Na+ absorption and their regulation could not be determined, because both isoforms are expressed by intestinal villus cells and are equally sensitive to amiloride inhibition. Thus regulation of these cloned isoforms by various stimuli was initially investigated in transfected nonepithelial cell lines (e.g., Refs. 13, 20, 21, 23, 27). A few studies, however, characterized their regulation in polarized epithelial cell lines, mostly renal ones (e.g., Refs. 2, 3, 30-32). However, the regulation of the cloned isoforms in intestinal cell lines has not been rigorously studied. Although the nonepithelial systems provided an opportunity to study individual isoforms without the confounding effects of other coexpressed NHEs, the observed functional characteristics of transfected NHE2 and NHE3 often differed substantially from the physiological properties of apical intestinal NHEs (1, 8, 14, 20, 21, 23, 27). For instance, these isoforms exhibit little or no basal activity and could only be studied after acid loading, a nonphysiological precondition. Furthermore, increases in cytosolic Ca2+ and cyclic nucleotides or activation of the PKC pathway often caused stimulation or little response. Finally, because these cell systems lack polarity, issues related to proper sorting and vectorial transport could not be addressed.

In light of these inherent problems, we chose to examine the regulation of NHE2 and NHE3 stably transfected in C2/bbe (C2) intestinal epithelial cells (28). The C2 cell is a subclone derived from a heterogeneous population of human Caco-2 intestinal epithelial cells. When grown on permeable supports, they form a confluent, electrically resistive (~200-300 Omega  · cm2) monolayer of polarized cells. The monolayers undergo spontaneous differentiation, as determined by the development of microvilli and markers of intestinal epithelial cell differentiation. By measurement of linear rates of 22Na+ unidirectional influxes from the luminal to the cellular and from the serosal to the cellular compartments of C2 postconfluent monolayers, it is possible to characterize the NHE activities of apical and basolateral membranes, respectively. Furthermore, by taking advantage of the differential sensitivities of NHE2 and NHE3 to the amiloride analog HOE-642 (13), it is possible to determine the contribution of each to total membrane NHE activity. This has been confirmed by several other laboratories (10, 13) as well as ours.

In this study, we show that both the function and the regulation of NHE2 and NHE3, when expressed in intestinal epithelial cells, differ from previous reports of their characterization in nonepithelial cell types but correlate with endogenous apical membrane NHE activity. On the basis of these observations, we believe the physiological properties of NHE2 and NHE3 may be dependent on epithelium-specific factors and/or conditional requirements.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials. Phenylmethylsulfonyl fluoride, aprotinin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). High-glucose DMEM, penicillin-streptomycin, and transferrin were purchased from Life Technologies (GIBCO BRL). Tissue culture dishes and Transwells were from Corning/Costar (Corning, NY). 22NaCl (1,000 Ci/g) and [3H]mannitol (22.5 Ci/mmol) were purchased from DuPont NEN. The amiloride analog 5-(N,N-dimethyl)amiloride (DMA) was a gift of Dr. Thomas Kleyman (University of Pennsylvania, Philadelphia, PA). 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphate (CPT-cGMP), and thapsigargin were from Alexis Biochemical (San Diego, CA), FCS was from Summit Biotech (Ft. Collins, CO), and anti-villin mouse IgG was from Transduction Laboratories (Lexington, KY). HOE-642 was a gift from Drs. Wolfgang Scholz and Hans-Jochen Lang (Hoechst, Frankfurt am Main, Germany).

Cell culture. C2 intestinal epithelial cells, generously provided by Dr. Mark Mooseker (Yale University, New Haven, CT), were grown as confluent monolayers on rat tail collagen-coated Transwells in DMEM supplemented with 10% FCS, 2 mM glutamine, 10 µg/ml transferrin, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of air containing 5% CO2. Differentiation of C2 cells in culture was determined by expression of villin and sucrase-isomaltase activities (Fig. 1, A and B).


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Fig. 1.   Phenotypic maturation of C2/bbe (C2) monolayers. A: C2 cells were plated on Transwells, and cells were harvested 2, 7, 14, and 21 days later. Cell protein was measured (by Bradford assay), and 10 µg of total cell protein were analyzed by a Western blot using a mouse monoclonal anti-human villin. Image is representative of 3 different experiments. B: proteins, harvested for villin in A, were also analyzed for enzymatic activity of the differentiation markers sucrase and alkaline phosphatase (Alk.P; µM · min-1 · mg protein-1). Enzyme activities were measured spectrophotometrically, as previously described (25), and specific activities are represented as nM · min-1 · mg protein-1.

Plasmid construction and purification of NHE2 and NHE3 fusion proteins. Fusion proteins were constructed for the predicted external loop of the fifth intermembranous domain of NHE2 (2M5b-M6 amino acids 260-280) (7) in pGEX-KT (18). A carboxy-terminal portion of NHE3 (amino acids 528-648 in pGEX-3X) was made as previously described (4). NHE1, NHE2, and NHE3 cDNAs were generously provided by Dr. Gary Shull (University of Cincinnati, Cincinnati, OH). Antibodies to these fusion proteins were produced as described (4).

Sucrase assay. Sucrase activity was measured in samples from 14-day-old Transwell cultures. Proteins were harvested by scraping and homogenized as for villin; however, no SDS-stop solution was added. Cell homogenates were diluted with 0.1 M sodium maleate buffer, and sucrase activity was measured by the ultramicro method of Messer and Dahlqvist (25). Briefly, samples were incubated with 50 mM sucrose at 37°C for 60 min, and then the glucose generated was measured using glucose oxidase and reacted with o-dianisidine, and the absorbance was measured at 450 nm. Sucrase values were expressed as nanomoles of glucose generated per minute per milligram of protein.

Western blot analysis. Cellular membranes were isolated, separated on 7.5% SDS-PAGE, and transferred to nitrocellulose membranes (0.2 µm; Schleicher and Schuell, Keene, NH) or Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA) in 25 mM Tris (pH 8.8), 0.1% SDS, 192 mM glycine, and 20% methanol at 1 A for 60 min. Blots were blocked in 5% Blotto [5% wt/vol low-fat milk, 10 mM sodium phosphate (pH 7.4), 150 mM NaCl, 2 mM EDTA, and 0.2% wt/vol Nonidet P-40 (NP-40)] for 1 h at 4°C. Primary antibody was mixed in blocking buffer (1:1,000 rabbit serum anti-2M5 and 1:800 anti-3C) and incubated overnight at 4°C. Blots were washed twice for 15 min in 5% blocking buffer and then incubated in donkey anti-rabbit horseradish peroxidase-conjugated antibody (Amersham, Arlington Heights, IL; 1:5,000) for 1 h at room temperature in 1% wt/vol Blotto. The blot was washed five times for 5 min and once for 15 min in 1% blocking buffer. Hybridized proteins were visualized by enhanced chemiluminescence (ECL; Amersham or Pierce, Rockford, IL).

22Na+ uptake studies. C2 cells were grown on rat tail collagen-coated Transwells for 14 days before experimentation. Cells were treated with either 10-4 M CPT-cAMP, 10-4 M CPT-cGMP, 10-7 M PMA, 1 µg/ml thapsigargin, hyperosmotic challenge (total osmolarity increased to 490 mosM with the addition of 210 mM mannitol), or 10% FCS for 15 min. Unidirectional apical membrane Na+ uptake (lumen to cell) was measured in flux buffer [in mM: 130 choline chloride, 5 KCl, 1 MgCl2, 2 CaCl2, 1 ouabain, 15 HEPES (pH 7.4), and 20 NaCl] with 1 µCi/ml 22NaCl for 10 min (during the linear uptake phase for 22Na+ influx in C2 cells of <15 min). Uptake was stopped after 10 min by four washes in cold wash buffer [in mM: 140 NaCl, 5 KCl, 15 HEPES (pH 7.4), and 1 sodium phosphate]. Cells were then solubilized in 1% SDS to measure intracellular Na+. Na+ influx (nmol) was calculated by dividing the average (liquid scintillation) dpm by the specific activity of the 22Na+ normalized to concentration of Na+ in the medium. The data are presented as the DMA (500 µM)-inhibitable component per Transwell, as previously described. HOE-642 was used to distinguish NHE2 (HOE-642-sensitive) from NHE3 (HOE-642-insensitive) activity. [3H]mannitol (1 µCi/ml) was used to correct for extracellular space (5).

Fluxes were also measured following acidification under acid-loaded conditions as previously described (6). Cells were incubated at room temperature for 60 min in acidifying saline consisting of (in mM) 50 NH4Cl, 70 choline chloride, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 15 HEPES (pH 7.0). Experimental additions of 8-chlorophenylthio-cAMP (50 µM), thapsigargin (50 ng/ml), or PMA (50 nM) were made during the last 10 min. Cells were washed once in acidifying saline with 120 mM choline chloride and fluxed for 10 min in flux buffer. To inhibit NHE2, 30 µM HOE-642 was added to the flux buffer. Both NHE2 and NHE3 were inhibited in 500 µM DMA.

Basolateral fluxes were measured under identical conditions, except that isotope was placed in the basolateral medium. Fluxes under basal and acid-loaded conditions were initiated and stopped identically.

Stable cDNA transfection of C2 cells. C2 cells were transfected with Lipofectin (Life Technologies) according to the manufacturer's suggested protocol and selected by resistance to G418 (Life Technologies), and expression was verified by Western blot analyses. The construction of the NHE3 cDNA expression vector is described in Bookstein et al. (4). Rat full-length NHE2 was obtained from G. Shull (36). Xba I and Kpn I restriction sites were added to the NHE2 cDNA at base 51 to facilitate subcloning. The pCN2 construct (pCB6+NHE2 cDNA) carried the full-length cDNA from Kpn I (bases 52-3689) and was subcloned into the expression vector pCB6+ (courtesy of J. Stinski). Clonal populations were then analyzed for NHE2 mRNA using isoform-specific probes and by Western blot using NHE2-specific antibodies.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization and validation of C2 cells as a viable cell model for the study of the intestinal brush-border NHEs. C2 cells were grown on collagen-coated Transwells to maximize differentiation and to facilitate a system for the study of apical vs. basolateral membrane Na+ uptake by NHEs. Because intestinal NHE2 and NHE3 are exclusively expressed by mature villus cells (4, 7, 15, 19), we wished to determine when the C2 cells, grown under our conditions, had fully differentiated. Prior studies on the differentiation of C2 cells (28) were done on cells grown on plastic or other permeable supports several weeks later than in our studies. Confluent cells, grown under our conditions, spontaneously differentiate over a 2-wk period (postplating at confluent numbers), as determined by increasing expression of villin (Fig. 1A) as well as increases in activities of the brush-border hydrolases, alkaline phosphatase, and sucrase-isomaltase (Fig. 1B and Ref. 25) and maturation of the apical microvillus membrane (data not shown). To confirm that the differentiation pattern did not differ in the NHE2- and NHE3-transfected cells, the well-established marker of differentiation in C2 cells, sucrase, was measured. Two days postplating, the level of sucrase (in nmol · min-1 · mg protein-1) did not differ among C2 (7.5 ± 0.6), NHE2-transfected C2 (C2N2; 7.3 ± 0.8), and NHE3-transfected C2 (C2N3; 8.1 ± 0.9) cells. After 14 days, the sucrase levels had increased to 72.2 ± 3.4 (C2), 66.9 ± 4.4 (C2N2), and 75.6 ± 7.3 (C2N3) nmol · min-1 · mg protein-1. The sucrase levels at day 14 did not differ among the three cell lines, suggesting that equivalent rates of brush-border maturation were occurring. All subsequent studies of transfected NHE2 and NHE3 were performed on fully differentiated C2 cells 14 days postplating.

Endogenous NHE expression in C2 cells. Apical and basolateral membrane NHE activities were measured during the linear phases of lumen-to-cell and serosa-to-cell unidirectional 22Na+ influxes, respectively, and are defined as the DMA-inhibitable component of Na+ influx. NHE activity is the difference between total and amiloride-insensitive influx. Differentiated C2 cells exhibit little or no basolateral membrane antiporter activity under basal conditions (Table 1), characteristic of the "housekeeping" antiporter NHE1. This basolateral flux is not altered in NHE2- or NHE3-transfected cells, demonstrating that these cells probably target these proteins to the apical membrane or maintain the active form only in the brush border. To determine whether the presence of an acid gradient would increase basolateral flux, as would be expected for NHE1, all three cell types (C2, C2N2, and C2N3) were acidified using a prepulse of ammonium saline. On removal of the ammonium ion, cellular acidification occurs as ammonia leaves the cell, leaving a proton behind. This manipulation activated the basolateral Na+ influx in all three cell types to a similar magnitude (Table 1). As shown in Table 1, under basal (non-acid-loaded) conditions, basolateral NHE activity significantly increased when intracellular Ca2+ was increased (via thapsigargin) or when PMA was present and was unchanged with CPT-cAMP. When the cells were acid loaded, basolateral NHE activity increased significantly but no further increase could be seen with the addition of thapsigargin or PMA. Treatment with Ca2+ or phorbol esters was shown previously to have no effect on the basolateral NHE in Caco-2 cells, but they were examined only under acid load (37). It is possible that acid loading maximally activates this exchanger and that NHE activity cannot be stimulated further. These findings underscore the importance of examining NHE activities under non-acid-loaded conditions. Furthermore, these results should be compared with those presented for apical membrane Na+ influxes, in which responses to various stimuli are significantly different. These data also indicate that C2 cells have the cellular machinery to respond to these second messenger pathways.

                              
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Table 1.   Effect of acid loading on second messenger inhibition of 22Na+ basolateral fluxes

In contrast to the absence of basolateral membrane NHE activity under basal (non-acid-loaded) conditions, C2 cells exhibit a small amount of endogenous brush-border membrane NHE activity (2.2 ± 0.2 nmol Na+ flux/Transwell, n = 6). By Western blot analysis, this activity appears to represent NHE2 (Fig. 2A). No endogenous NHE3 is expressed by C2 cells under the conditions used in this study (Fig. 2B).


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Fig. 2.   Western blot verification of expression of Na+/H+ exchanger (NHE) 2 and NHE3 in transfected C2 cells. Left: NHE2-transfected C2 (C2N2) and C2 cells were examined for presence of NHE2 in contrast to control PS120 cells. All lanes were equally loaded with 60 µg membrane protein/lane. Right: NHE3-transfected C2 (C2N3) and C2 cells were examined for presence of NHE3 by Western blot analysis as described in EXPERIMENTAL PROCEDURES.

Expression and function of NHE2 and NHE3 in stably transfected C2 cells. C2 cells were transfected with either rat NHE2 (C2N2) or NHE3 (C2N3). As shown by Western blot analyses in Fig. 2, left and right, transfected C2 cells appropriately express NHE2 and NHE3 protein, respectively. The differentiated phenotype for apical membrane NHE activation was examined in the presence of various second messenger regulators of exchanger function. Unidirectional influx studies presented here (see Figs. 4 and 5) demonstrate apical membrane localization of NHE2 and NHE3, analogous to the membrane location of the endogenously expressed isoforms in intestinal epithelium (4, 19).

Western blot analyses demonstrated significantly increased NHE2 protein expression in C2N2 when contrasted to nontransfected cells (Fig. 2, left). This increase in expression of NHE2 was also verified by a corresponding increase in basal levels of apical membrane NHE2 activity (~3-fold) over that of nontransfected control C2 cells, without alterations in basolateral activity. At 20 mM external Na+, NHE2 exhibited an apical basal net Na+ influx of 6.7 ± 0.8 nmol/Transwell under control conditions (see Fig. 4).

HOE-642 can be used to distinguish between the activities of NHE2 and NHE3 in C2 cells, as previously reported (10, 13). At 30 µM, HOE-642 inhibited ~87% of NHE2 activity but only 13% of NHE3 activity (Fig. 3). In contrast, when C2N3 cells were examined in the presence of 30 µM HOE-642, minimal inhibition of apical membrane NHE activity was observed (Fig. 3). Thus the differential sensitivities of NHE2 and NHE3 to HOE-642 can be used to discriminate between the activities of these isoforms.


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Fig. 3.   HOE-642 dose-response curve in C2N2 and C2N3 cells. HOE-642-sensitive Na+ influx was measured in C2N2 cells at 0.01, 1, 3, 10, 30, and 100 µM and in C2N3 cells at 0.01, 3, 10, 100, 300 and 1,000 µM (n = 3; means ± SE). On y-axis, activity is presented as percent of total activity in absence of HOE-642; x-axis shows increasing concentrations of HOE-642 on a log scale. At 30 µM HOE-642, NHE2 was inhibited by ~87% in C2N2 cells and NHE3 was inhibited by ~13% in C2N3 cells.

Regulation of NHE2 and NHE3 in the intestinal epithelial C2 cell. The effects of a number of regulators on apical membrane NHE2 activity were examined next (Fig. 4). NHE2 was activated by serum (14.0 ± 1.1 vs. a basal value of 6.7 ± 0.8 nmol/Transwell) but was unaffected by activation of PKC-dependent pathways (via PMA; 6.8 ± 0.8 nmol Na/Transwell) or cGMP-dependent protein kinase (PKG)-dependent pathways (via CPT-cGMP; 6.8 ± 0.9 nmol Na/Transwell). Thapsigargin, a non-phorbol ester type tumor promoter, inhibits microsomal Ca2+-ATPase, elevating intracellular Ca2+ and activating mitogen-activated protein kinase independently of PKC or Ca2+ influx (9). NHE2 was inhibited by thapsigargin (1.0 ± 0.5 nmol Na/Transwell) and cAMP (2.9 ± 0.7 nmol Na/Transwell)-dependent pathways and by hypertonicity (2.1 ± 0.3 nmol Na/Transwell). These results are in marked contrast to regulation by CPT-cAMP or PKC-dependent pathways and by hypertonicity in NHE2-transfected nonepithelial cells (20, 21).


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Fig. 4.   Apical membrane Na+ uptake in C2N2 cells. 5-(N,N-dimethyl)amiloride (DMA)-inhibitable 22Na+ influx was measured in non-acid-loaded C2N2 cells, as described in EXPERIMENTAL PROCEDURES. Unidirectional Na+ uptake was measured for 10 min at 20 mM NaCl following 15 min under control conditions (n = 6) or 15 min of treatment with 1 µg/ml thapsigargin (Thaps; n = 3), 10-7 M phorbol 12-myristate 13-acetate (PMA; n = 3), 10-4 M CPT-cAMP (cAMP; n = 4), 10-4 M CPT-cGMP (cGMP; n = 4), 10% FCS (Serum; n = 3), or osmotic stress (490 mosM; Hyper; n = 3).

A significant increase in apical membrane NHE activity compared with nontransfected control cells was also observed in C2N3 cells. As before, experiments presented here were performed under basal conditions, without acid loading. At 20 mM external Na+, the basal unstimulated Na+ flux in C2N3 cells was greater than that observed in C2 or C2N2 cells (Figs. 4 and 5). Examination of NHE3 regulation in C2N3 cells (Fig. 5) demonstrated that NHE3 activity was inhibited by PMA (3.3 ± 0.7 nmol Na/Transwell), cyclic nucleotides (cAMP 4.4 ± 1.6 nmol Na/Transwell and cGMP 2.6 ± 0.5 nmol Na/Transwell), and Ca2+-dependent pathways (2.4 ± 0.7 nmol Na/Transwell) compared with basal levels of 16.1 ± 1.5 nmol Na/Transwell. The addition of 10% serum 10 min before flux caused a small but statistically significant inhibition (13.0 ± 1.5 nmol Na/Transwell, P < 0.05). Hypertonicity (490 mosM) also caused a significant inhibition of NHE3 activity under basal conditions (3.0 ± 0.9 nmol Na/Transwell; data not shown).


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Fig. 5.   DMA- and HOE-642-inhibitable Na+ influx in C2N3 cells. DMA-inhibitable and HOE-642-inhibitable unidirectional 22Na+ influxes from apical membranes were measured in C2N3 cells. Non-acid-loaded cells were treated for 15 min with 1 µg/ml thapsigargin (DMA, n = 9; HOE-642, n = 4), 10-7 M PMA (DMA, n = 10; HOE-642, n = 4), 10-4 M CPT-cAMP (DMA, n = 9; HOE-642, n = 4), 10-4 M CPT-cGMP (DMA, n = 8; HOE-642, n = 4), 10% FCS (DMA, n = 9; HOE-642, n = 4), or osmotic stress (490 mosM; DMA, n = 5, not shown). Control received no additions (DMA, n = 16; HOE-642, n = 4). Difference between DMA- and HOE-642-inhibitable components represents NHE3 activity.

NHE3 activity was unaffected by the presence of endogenous NHE2 activity. The difference between the DMA- and HOE-642-inhibitable Na+ fluxes (Fig. 5) represents NHE3 activity only. In fact, NHE3 activities examined in the presence and absence of HOE-642 were virtually identical, suggesting that the presence of NHE2 does not affect the regulation of NHE3. Clearly, NHE3-specific regulation can be examined in C2 cells in the presence or absence of NHE2.

The observed responses of apical membrane NHE2 and NHE3 to various stimuli in C2 cells differ from their responses when they are expressed in nonepithelial, nonintestinal cell types (20, 21, 23, 27, 33-35, 39, 41). This could be due to the presence of cell-specific modifying factors or to a secondary change in the intracellular condition of the cell that would inhibit NHE activity independent of the agent used. The most obvious alteration to consider would be stimulated increases in intracellular pH that could diminish the plasma membrane pH gradient and secondarily inhibit Na+/H+ exchange activity. To address this issue, cells were acid loaded and treated with CPT-cAMP, thapsigargin, or PMA for the last 10 min of the 30-min exposure to ammonium saline. 22Na+ unidirectional flux measurements across the apical membrane were performed immediately after removal of ammonium saline (with only one <2-s wash in isotonic choline chloride). As shown in Table 2, acidification significantly increased apical influx of 22Na+ in C2, C2N2, and C2N3 cells. Nevertheless, the pattern of response to these second messengers was identical to that observed under basal conditions, suggesting that the pH status of the cell did not alter regulatory mechanisms. This could be due to cell-specific modification of the apical NHEs, alterations in membrane trafficking, and/or modulation of an NHE-associated regulatory protein.

                              
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Table 2.   Effect of acid loading on second messenger inhibition of 22Na+ apical influxes

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Before the present studies, the function and regulation of the apical, epithelium-specific NHE isoforms NHE2 and NHE3 had been most widely characterized in transfected, nonepithelial cell lines such as PS120 (Chinese hamster lung fibroblasts) and AP-1 (Chinese hamster ovary) (20, 21, 23, 27, 39). Although these studies demonstrated important functional and regulatory differences between the NHE isoforms, several findings contrast with previous observations of apical membrane NHE activity made in vivo. It should be noted that a number of studies of NHE activity in polarized renal cell lines have been conducted (e.g., Refs. 2, 3, 11, 30-32). Although definitive characterization of the isoform present was not always confirmed, these studies have been useful in demonstrating the merits of using epithelial cell systems to examine NHE2 and NHE3. Analysis of NHE2 and NHE3 regulation in C2 cells presented here validates the C2 line as a viable model for regulation in fully differentiated intestinal epithelium and demonstrates the importance of cell-specific factors in understanding regulation in vivo. Furthermore, we show that NHE2-specific regulation can be analyzed in the presence of active NHE3.

The activities of NHE2 and NHE3 expressed in PS120 and AP-1 cells could only be demonstrated under acid-loaded conditions and not under basal conditions. Although others have selected acid loading as a mechanism to achieve maximal activation of antiporter function to more effectively examine kinetic parameters, we chose to examine activities under non-acid-loaded conditions, which is more representative of the physiological condition. For example, intact epithelial cells, whether studied in vivo or in vitro, have apical NHE activity that can be demonstrated under non-acid-loaded conditions (1, 8, 14, 17, 24, 29). Studies done by Semrad et al. (29) demonstrated that Na+ absorption by brush-border membrane NHEs in enterocytes existed at an ongoing basal level in the absence of specific stimulation. This is teleologically important, as NHE2 and NHE3 in vivo must be prepared to mediate vectorial Na+ transport when presented with a luminal salt load. Another reason for examining NHE2 and NHE3 under these conditions is that the regulation of these isoforms in nonepithelial cell lines has not always correlated with observations made in the gut. Also, as demonstrated by the response of the basolateral C2 NHE to thapsigargin and PMA without acid load (Table 1), acid loading itself can mask regulatory responses.

Our examination of the regulation of apical membrane antiporter function in C2 intestinal cells underscores the disparities between NHE2 and NHE3 regulation in transfected nonepithelial cells and regulation observed in vivo. The studies done to date on intact intestinal brush borders have had to rely on amiloride analogs to identify NHE activity. Because these do not distinguish NHE2 from NHE3 activity, we can only compare our results to reported total brush-border NHE activity for the in vivo studies. For instance, in studies of NHE function in the intestinal epithelium, inhibition of brush-border membrane Na+ absorption has been shown to be mediated by PKC-, cGMP-, and Ca2+-dependent pathways (1, 8, 14). Ileal mucosal NHE activity was also inhibited by PKG via a Ca2+-dependent pathway (29). Guandalini et al. (17) showed that the activation of guanylate cyclase by a heat-stable enterotoxin resulted in inhibition of Na+ influx at the apical surface of the intestine. NHE2 in C2 cells was unaffected by increases in cGMP, whereas NHE3 was inhibited; neither isoform in PS120 fibroblasts was affected by this nucleotide (23). Increases in cGMP had no effect on NHE1, NHE2, or NHE3 activities in the AP-1 cells (20). Ca2+/calmodulin and ATP inhibited NHE activity on the brush-border membrane vesicles of rabbit small intestine (12, 16), and Donowitz et al. (14) demonstrated that an elevation in Ca2+ inhibited rabbit ileal Na+ absorption via a PKC pathway. In C2 cells, increases in cytosolic Ca2+ via the non-phorbol ester type tumor promoter thapsigargin inhibited both NHE2 and NHE3. Ahn et al. (1) demonstrated that ongoing basal Na+ uptake, due to NHE activity, from the luminal surface of rabbit proximal colon was inhibited by phorbol diesters. PMA alone did not alter NHE2 activity in C2 cells, whereas NHE3 was almost completely inactivated. Serum, on the other hand, caused a dramatic stimulation of NHE2 and a modest decrease of NHE3 activity. Recent studies identified cAMP-dependent regulatory sites on NHE3 (22), as well as regulatory proteins (38, 40) that may be involved in inhibiting NaCl absorption in the gut in response to toxins. Whereas both NHE2 and NHE3 activities in C2 intestinal epithelial cells were inhibited by increases in cAMP (Figs. 4 and 5), neither isoform in PS120 fibroblasts (23) exhibited any regulation by cAMP. In transfected AP-1 cells, cAMP-dependent protein kinase A stimulated NHE2 but decreased the acid-stimulated activity of NHE3 (20, 21). Another difference noted was the effect of hypertonicity on the brush-border NHEs. In PS120 cells, both NHE2 and NHE3 were inhibited by hyperosmotic stress (41); in AP-1 cells, only NHE3 was inhibited (21). In C2 cells, both isoforms were inhibited.

These data underline the importance of cell-specific factors or conditions that critically determine the functional and regulatory properties of NHE2 and NHE3. As we demonstrate here, regulation of NHE2 and NHE3 in C2 cells closely resembles previous studies of intestinal epithelial brush-border activities. Recently, regulatory proteins have been identified that control NHE3 function following specific stimuli. For example, two proteins, NHERF (38) and E3KARP (40), have been identified that mediate cAMP-stimulated inhibition of NHE3 in renal and intestinal systems, respectively. Reportedly, the Caco-2 cell line does not possess E3KARP (40). C2 cells, being a subclone of the Caco-2 cells, may express this protein at levels that would be "masked" in the "parental" population because they represent a minor subset of the Caco-2 population. Alternatively, another as yet unidentified regulatory protein may fill this role in C2 cells. To date, there have been no reports that similar regulatory proteins and/or mechanisms of regulation exist for NHE2.

Our data support the hypothesis that NHE3 can be expressed in the same cells as NHE2 and yet be regulated independently (Fig. 5). This suggests a unique role for NHE2 in the gut. As proposed by Maher et al. (24), although NHE3 may be the primary NHE responsible for vectorial Na+ transport, under special conditions that would necessitate a modification in Na+ absorption mediated by the inactivation of NHE3, NHE2 may be required to function in its place. Another possible role for this unique exchanger, suggested by its dramatic response to serum, could be in the process of development or repair of the gut epithelium.

C2 cells are ideally suited for the study of apical membrane NHE isoforms, as they have little basal, or unstimulated, endogenous brush-border NHE activity. As in mature enterocytes, our studies in C2 cells demonstrate that NHE2 and NHE3 activities are localized exclusively to the apical membrane domain (cf. Tables 1 and 2), an observation previously made in wild-type Caco-2 cells (33-35). Unidirectional 22Na+ influx measurements in C2N2 cells demonstrated an approximately threefold enhancement of uptake of Na+ over nontransfected cells under basal unstimulated conditions, with no increase in basolateral flux (Table 1). Apical membrane Na+ uptake in C2N3 cells was enhanced approximately sevenfold over the level in nontransfected parental cells, also with no increase in basolateral flux (Table 1). These data indicate that the transfected C2 cells mimic the phenotype of enterocytes and exhibit a similar pattern of brush-border NHE regulation, underscoring their utility as a cell model for examination of the function of NHE2 and NHE3 in the gut (4, 7, 15, 19, 24). This also suggests that the brush-border NHEs are targeted to the apical membrane exclusively or are active only in the apical domain. It should be noted that these cells have not been transfected with NHE1. The C2 cells possess native basolateral activity that resembles NHE1 in regulation (Table 1). Because the goal of the present studies was to investigate the regulation of the apical isoforms, no characterization of the housekeeping NHE was undertaken. We do not know whether C2 cells would target NHE1 to both membrane domains, as has been demonstrated for the Caco-2, OK, and HT-29 cell lines (26). Our inhibitor concentrations do not rule out NHE1 as a minor component of NHE2 activity. In the Noel et al. study (26), the cell lines used to look at NHE3 sorting, NHE3-transfected OK and Madin-Darby canine kidney cells, sorted this brush-border isoform exclusively to the apical membrane.

We measured NHE activity by 22Na+ influx, a precise technique that specifically measures NHE function without the added complexity of measuring changes in intracellular pH, by which interpretations can be biased due to changes in the buffering capacity of the cell population. Our data indicate that the transfected C2 cells mimic the phenotype of enterocytes and exhibit a similar pattern of brush-border NHE regulation, underscoring their utility as a cell model for examination of the function of NHE2 and NHE3 in the gut.

Evidence presented here demonstrates unique differences in the regulation of NHE2 and NHE3, in contrast to previous studies in nonepithelial cell lines (20, 21, 23, 27, 37), but parallels and provides the foundation for understanding brush-border membrane Na+ absorptive properties of intact intestinal epithelial cells (1, 8, 10, 14, 17, 24). Furthermore, these studies validate the utility of the C2 cell line as a cell model for examination of the functional roles and biochemical regulation of brush-border membrane NHE isoforms NHE2 and NHE3 in the gut. The homogeneous phenotype of the C2 cell line provides a better experimental model than the heterogeneous Caco-2 cells because Caco-2 exhibit no endogenous apical membrane NHE activity under basal conditions and because activation of transfected apical membrane NHE in Caco-2 was only demonstrated after acid loading (33-35, 37). Such artificial conditions may not reflect normal physiology. This further supports the appropriateness of the C2 cell line, in which we have shown that, as in the gut (1, 8, 10, 17, 24), apical membrane antiporter activity occurs under basal conditions. In conclusion, our studies demonstrate that C2 cells provide a powerful tool to examine the function of NHE2 and NHE3 in enterocytes.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38510 and Digestive Disease Core Grant DK-42086 and by the Gastrointestinal Research Foundation of Chicago.

    FOOTNOTES

Address for reprint requests: E. B. Chang, Dept. of Medicine, MC6084, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637.

Received 16 October 1997; accepted in final form 28 May 1998.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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