Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon

Mutsuhiro Ikuma1, Michael Kashgarian2, Henry J. Binder1, and Vazhaikkurichi M. Rajendran1

Departments of 1 Internal Medicine and 2 Pathology, Yale University School of Medicine, New Haven, Connecticut 06520


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Dietary sodium depletion has multiple diverse effects on ion transport in the rat colon, including both the induction and inhibition of electroneutral NaCl absorption in proximal and distal colon of rat, respectively. To establish the mechanism of the differential regulation of Na+ absorption by sodium depletion, this study utilized 1) HOE-694, a dose-dependent inhibitor of Na+/H+ exchanger (NHE) isoforms, in studies of proton gradient-driven 22Na uptake (i.e., Na+/H+ exchange) by apical membrane vesicles (AMV); 2) Northern blot analyses of NHE isoform-specific mRNA abundance; and 3) Western blot analyses of NHE isoform-specific protein expression. HOE-694 inhibition studies establish that 25 µM HOE-694-sensitive (NHE2) and 25 µM HOE-694-insensitive (NHE3) Na+/H+ exchange activities are present in AMV of both proximal and distal colon of normal rats. In proximal colon, dietary sodium depletion enhanced both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance. In contrast, in distal colon both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance were inhibited by sodium depletion. NHE1 isoform-specific mRNA abundance in proximal or distal colon was not altered by sodium depletion. Differential effects by sodium depletion on Na+/H+ exchange in rat colon are tissue specific and isoform specific; sodium depletion both induces and inhibits apical Na+/H+ exchange at a pretranslational level.

aldosterone; pretranslational; Na+/H+ exchange; membrane vesicles; uptake


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE MECHANISMS OF ACTIVE NaCl transport differ significantly in the proximal and distal segments of the rat large intestine (4). HCO-3-dependent electroneutral NaCl absorption is the primary transport process in the distal colon and is the consequence of the coupling of parallel ion exchanges, Na+/H+ and Cl-/HCO-3 exchanges (3, 14, 16, 23, 24). Although electroneutral Na+ absorption is also Cl- dependent in the proximal colon, net Cl- movement is absent (13), and Na+/H+ exchange is the predominant mechanism of Na+ absorption (13, 23).

Aldosterone, either as a consequence of dietary sodium depletion or as a result of its continuous infusion via minipumps,1 alters both Na+ and Cl- absorption in both proximal and distal segments of the rat colon (15, 18). However, the effect of aldosterone in these two segments differs substantially: aldosterone inhibits electroneutral NaCl absorption in the distal colon; in contrast, in the proximal segment aldosterone enhances Na+ absorption while inducing Cl- absorption (15, 18).

Five different Na+/H+ exchange isoforms have been cloned to date, and three isoforms (NHE1, NHE2, and NHE3) have been identified in intestinal epithelial cells (12, 17, 21, 32). Recently, a Cl--dependent Na+/H+ exchange has been identified in apical membranes of crypt cells of rat distal colon (25), and it is not known whether any of the existing NHE isoforms are responsible for Cl--dependent Na+/H+ exchange activity.

The molecular regulation of colonic Na+/H+ exchange by aldosterone has recently been reported by Cho et al. (7). These studies examined the effect of aldosterone administrated intraperitoneally for only 3 days and concluded that aldosterone did not alter any parameter of Na+/H+ exchange in the distal colon while stimulating NHE3, but not NHE2, isoform-specific message, protein, and transport function in the proximal colon (7). We had previously observed that 3 days of continuous subcutaneous infusion of aldosterone did not modify electroneutral NaCl absorption in the distal colon, whereas 7 days resulted in its complete inhibition (18, 30). Because intraperitoneal administration of aldosterone for 3 days resulted in serum aldosterone levels that are 24% less than those observed in dietary sodium depletion, a physiological model of aldosterone excess, the present study was designed to provide a comprehensive characterization of the effects of dietary sodium depletion1 on Na+/H+ exchange in both proximal and distal segments of the rat colon.

The present results establish that sodium depletion alters both NHE2 and NHE3 isoforms at a pretranslational level in both proximal and distal segments; sodium depletion substantially increases both NHE2 and NHE3 function, protein, and message in the proximal colon but reduces NHE2 and NHE3 function, protein, and message in distal colon. In contrast, sodium depletion does not alter NHE1 isoform-specific function or message. Thus the differential effects of sodium depletion on Na+/H+ exchange are both isoform specific and tissue specific.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Membrane Vesicle Preparation

Apical membrane vesicles (AMV) and basolateral membrane vesicles (BLMV) were isolated from proximal and distal segments of the large intestine of both normal and aldosterone-treated rats (male, Sprague Dawley, 200-250 g) by the method of Stieger et al. (29) and Biber et al. (1), respectively, as described previously (23, 26, 27). Normal rats were fed with Purina rat chow ad libitum, whereas aldosterone rats were produced by feeding Na+-free diet (20 g · rat-1 · day-1) prepared in our laboratory for 7 days, as described previously (13).1 Purity of AMV (10- to 12-fold) and BLMV (16- to 18-fold) were assessed by K+-ATPase and Na+-K+-ATPase activities enrichment, respectively (10, 17). Protein was measured by the method of Lowry et al. (20).

Uptake Studies

Uptake of 22Na was performed for 6 s by the rapid filtration techniques, as both proton gradient-driven and potential-dependent 22Na uptake were linear for at least up to 8 s (23, 26, 27). Uptake studies were performed in at least three different membrane preparations. Results presented represent means ± SE of triplicate assays of typical experiments.

mRNA Isolation

Total RNA from colonic epithelial cells were isolated by the CsCl cushioning centrifugation method of Sambrook et al. (28). In brief, proximal and distal colonic segments obtained from anesthetized rats were washed with ice-cold saline containing 0.5 mM dithiothreitol (DTT). Sacs of colonic segments filled with buffer A (4 mM HEPES-Tris, pH 7.6, 30 mM NaCl, 2 mM EDTA, and 0.5 mM DTT) were incubated in buffer B (buffer A without DTT) on ice. After 20-30 min of incubation, colonocytes were squeezed directly into 15-ml GIT buffer (4 M guanidineisothiocyanate, 25 mM Na+-acetate, and 0.85% beta -mercaptoethanol) and homogenized. Homogenate was layered on CsCl buffer (5.7 M CsCl, 25 mM Na+-acetate, pH 6.0) in a Polyallomer tube and centrifuged at 32,000 rpm for 18 h at 20°C. Glassy RNA pellet was dissolved in TE buffer (10 mM Tris · HCl, 1 mM EDTA, pH 7.5). RNA samples were electrophoresed on 1% agarose-formaldehyde gel. RNAs with intact 28S and 18S ribosomal RNAs were used for mRNA purification by oligo(dT) (Boehringer-Mannheim, Indianapolis, IN) column.

cDNA Probes

NHE isoform-specific cDNA probes (NHE1: Pst I fragment that encodes nt 1237-2611; NHE2: Pvu II fragment that encodes nt 849-1590; and NHE3: Pst I fragment that encodes nt 1241-2522) were digested from their respective full-length cDNAs, generously provided by Dr. Gary E. Shull, University of Cincinnati, Cincinnati, OH (22, 33). Digestion products were electrophoresed on 1% agarose-ethidium bromide gel. Expected size cDNA fragments extracted using cDNA gel extraction kit (Qiagen, Chatsworth, CA) were used as NHE isoform-specific probes for Northern blot analyses. Glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was prepared using rat GAPDH control amplifier set (Clontech, Palo Alto, CA).

Northern Blot

Five micrograms mRNA in 50% formamide-15% formaldehyde were electrophoresed on a 1% agarose-formaldehyde gel. Electrophoresed RNA was transferred to nylon membrane (NEN, Boston, MA) by capillary action and linked to membrane using UV-Stratalinker 2400 (Stratagene, La Jolla, CA). Blots were exposed for 2 h in prehybridization solution (50% deionized formamide, 10% dextran sulfate, 1 M NaCl, and 1% SDS) at 42°C. cDNA probes were labeled using [32P]dCTP and random primer labeling kit (Boehringer-Mannheim). 32P-labeled cDNA probes were added and hybridized for 18 h at 42°C. The blots were washed with 0.1× saline-sodium citrate-0.5% SDS at 45°C for 30 min and exposed to Hyperfilm (Amersham, Chicago, IL) at -80°C, and the films were developed at various times. Individual blots were stripped and hybridized sequentially with each of the probes (NHE1, NHE2, NHE3, and GAPDH). NHE isoform-specific mRNA abundance was quantitated with Personal Densitometer SI using ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA) and normalized to GAPDH mRNA level.

Western Blot

SDS-PAGE was performed by the standard protocol as described earlier (26). To avoid aggregation, the protein samples (30 mg) of rat colonic apical and basolateral membranes were warmed to 37°C for 20 min before loading. Proteins were electrophoretically transferred from SDS-PAGE to nitrocellulose (Biotrace; Gelman Sciences, Ann Arbor, MI) in 192 mM glycine, 25 mM Tris, pH 8.3, and 20% methanol overnight at 30 V. After nonspecific sites were blocked with TBST consisting of 20 mM Tris, 137 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk, pH 7.5, immunostaining was performed with NHE2 and NHE3 isoform-specific antibodies (2, 19) and horseradish peroxidase-conjugated anti-rabbit IgG (Amersham) and anti-mouse IgG (Amersham), respectively. Antibody-specific bands were visualized by Supersignal enhanced chemiluminescence (Pierce Chemicals, Rockford, IL). Mouse monoclonal antibody against beta -actin (Sigma, St. Louis, MO) was used as constitutively expressed probe. NHE2 and NHE3 isoform-specific antibodies used in these Western blot analyses were kindly provided by Drs. Mark Donowitz (Johns Hopkins University School of Medicine, Baltimore, MD) and Daniel Biemesderfer (Yale University, New Haven, CT), respectively. NHE isoform-specific protein abundance was quantitated with Personal Densitometer SI using ImageQuant software.

Statistics

Results are reported as means ± SE. Student's t-test was used to determine statistical significance between two groups. For more than two-group comparison, a one-way ANOVA was performed. A P value of <0.05 was considered significant.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Proximal Colon

22Na uptake studies. Previous studies of transepithelial 22Na fluxes across intact tissue established that dietary sodium depletion and aldosterone enhanced active Na+ absorption in rat proximal colon (13, 31). Therefore, experiments were designed to examine the mechanisms of this induction of electroneutral Na+ absorption by sodium depletion in rat proximal colon. In the initial studies, electroneutral Na+/H+ exchange activity was measured as outward proton gradient-driven 22Na uptake in both AMV and BLMV isolated from proximal colon of both normal and sodium-depleted rats. As shown in Fig. 1, the initial rate of proton gradient-driven 22Na uptake in AMV prepared from normal proximal colon was almost identical to that of our earlier results (23). Proton gradient-driven 22Na uptake in AMV from proximal colon of experimental rats was substantially higher than that in AMV prepared from normal rat proximal colon. Figure 1A presents the results of 22Na uptake by AMV from normal and sodium-depleted rats and demonstrated a 2.6-fold enhancement in the experimental group. In contrast, the rate of proton gradient-driven 22Na uptake in BLMV was similar in the two groups (Fig. 1B). Proton gradient-driven 22Na uptake was inhibited by 5 µM 5-ethylisopropylamiloride (EIPA), an amiloride analog that inhibits Na+/H+ exchange, in AMV of normal (37.2 ± 6.5 vs. 0.3 ± 0.4 pmol · mg protein-1 · 6 s-1) and sodium-depleted (94.7 ± 2.6 vs. 0.0 ± 0.2 pmol · mg protein-1 · 6 s-1) rats. This observation confirms that proton gradient-driven 22Na uptake primarily represents Na+/H+ exchange in both normal and sodium-depleted animals. These results indicate that sodium depletion enhances electroneutral Na+ absorption in proximal colon by increasing apical, but not basolateral, membrane Na+/H+ exchange.


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Fig. 1.   Effect of aldosterone on proton gradient-driven 22Na uptake. Apical membrane vesicles (AMV; A) and basolateral membrane vesicles (BLMV; B) isolated from proximal colon of normal (open bars) and sodium-depleted (solid bars) rats were preloaded with 150 mM K+-gluconate and 50 mM Mes-Tris (pH 5.5). Uptake was measured for 6 s by incubating the vesicles in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). Uptake was also measured in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM Mes-Tris (pH 5.5). Proton gradient-driven uptake presented was calculated by subtracting the uptake obtained in medium with Mes-Tris (pH 5.5) from that in medium with HEPES-Tris (pH 7.5). All media contained 25 µM valinomycin and 0.8% ethanol. * P < 0.01.

Kinetic studies were performed to establish whether the increase in proton gradient-driven 22Na uptake in AMV from proximal colon of sodium-depleted rats is due to change in affinity (Km) for Na+ and/or the maximal rate (Vmax) of uptake. As shown in Fig. 2, increasing extravesicular Na+ concentration both increased and saturated proton gradient-driven 22Na uptake in AMV from proximal colon of both normal and experimental rats. Lineweaver-Burke plot analyses of proton gradient-driven 22Na uptake in AMV from proximal colon of normal and sodium-depleted rats yielded Km values for Na+ of 8.3 ± 0.3 and 9.7 ± 0.4 mM (not significant) and Vmax values of 4.8 ± 0.2 and 17.4 ± 5.7 nmol · mg protein-1 · 6 s-1 (P < 0.05), respectively (Fig. 2B). Significant increase in Vmax (3.6 fold) but not Km for Na+ of the proton gradient-driven 22Na uptake suggests that sodium depletion might have either enhanced the turnover rate of the existing Na+/H+ exchange or induced the synthesis of new Na+/H+ exchange proteins.


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Fig. 2.   Effect of Na+ concentrations on proton gradient-driven 22Na uptake. A: AMV isolated from proximal colon of normal (bullet ) and sodium-depleted (open circle ) rats were preloaded with 50 mM K+-gluconate, 100 mM N-methyl-D-glucamine (NMG)-gluconate, and 50 mM Mes-Tris (pH 5.5). Uptake was measured for 6 s by diluting the vesicles in medium containing 50 mM K+-gluconate, varying concentrations of Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). NMG-gluconate concentration was adjusted to maintain medium isosmolarity. Uptake was also measured in medium with 50 mM K+-gluconate, varying concentrations of Na+-gluconate, trace of 22Na, and 50 mM Mes-Tris (pH 5.5). Proton gradient-driven uptake presented in A was calculated by subtracting the uptake obtained in medium with Mes-Tris (pH 5.5) from that in medium with HEPES-Tris (pH 7.5). All media contained 25 µM valinomycin and 0.8% ethanol. B: Lineweaver-Burke transformation of data presented in A. Kinetic parameters calculated from 3 different identical experiments are Km for Na+ of 8.3 ± 0.3 and 9.7 ± 0.4 mM (not significant) and maximal rate (Vmax) of 4.8 ± 0.2 and 17.4 ± 5.7 nmol · mg protein-1 · 6 s-1 (P < 0.05) for normal and sodium-depleted rats, respectively.

Studies to distinguish NHE2 and NHE3 isoforms activities. Studies were designed to identify whether NHE2 and/or NHE3 isoforms are responsible for the enhancement of Na+/H+ exchange activity by sodium depletion in AMV from proximal colon. In these studies, HOE-694 (3-methylsulfonyl-4-piperidinobenzoyl guanidine), an amiloride analog that inhibits the functional expression of NHE isoforms in a dose-dependent manner (9, 35),2 was used to estimate the several components of proton gradient-driven 22Na uptake by specific NHE isoforms. In these experiments the effect of HOE-694 on Na+/H+ exchange was determined by assessing its ability to alter the EIPA-sensitive component of proton gradient-driven 22Na uptake. As shown in Fig. 3B, proton gradient-driven 22Na uptake in BLMV was inhibited by 1 µM HOE-694 in both normal and sodium-depleted groups. This fraction represents the NHE1 isoform and is ~96% of total Na+/H+ exchange function in BLMV. In contrast, proton gradient-driven 22Na uptake in AMV was not significantly inhibited by 1 µM HOE-694 in either group (Fig. 3A), confirming that NHE1 isoform is localized to basolateral, not apical, membranes and that sodium depletion does not alter the membrane localization of NHE1 isoform.


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Fig. 3.   Effect of 1 µM HOE-694 on proton gradient-driven 22Na uptake. AMV (A) and BLMV (B) isolated from proximal colon of normal and sodium-depleted rats were preloaded with 150 mM K+-gluconate and 50 mM Mes-Tris (pH 5.5). Uptake was measured for 6 s by incubating the vesicles in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). Uptake was also measured in the presence of either 5 µM 5-ethylisopropylamiloride (EIPA) or 1 µM HOE-694. EIPA-sensitive uptake was calculated by subtracting the uptake observed in the presence of EIPA from that in its absence and represents total Na+/H+ exchange activity (open bars). Solid bars represent Na+/H+ exchange in the presence of 1 µM HOE-694. The 1 µM HOE-694-sensitive component represents Na+/H+ exchanger isoform 1 (NHE1)-specific Na+/H+ exchange activity. All media contained 25 µM valinomycin and 0.8% ethanol. Aldo, aldosterone. * P < 0.01.

Figure 4 presents 25 µM HOE-694-sensitive and 25 µM HOE-694-insensitive proton gradient-driven 22Na uptake in AMV of both normal and sodium-depleted rats. These HOE-694-sensitive and HOE-694-insensitive components presented represent EIPA-sensitive proton gradient-driven 22Na uptake. On the basis of the inhibition of proton gradient-driven 22Na uptake by HOE-694, NHE3 is the predominant NHE isoform present in AMV. Sodium depletion induced both NHE2 isoform and NHE3 isoform activities, although not proportionately. As a result, NHE2 isoform represents 11% of total EIPA-sensitive proton gradient-driven 22Na uptake in normal rats, whereas in the experimental group NHE2 isoform represents 31% of EIPA-sensitive proton gradient-driven 22Na uptake. Thus sodium depletion increased the NHE2 isoform component of proton gradient-driven 22Na uptake by 7.6-fold, whereas the NHE3 isoform component was increased by only 2-fold. These results suggest that both NHE2 and NHE3 isoforms are responsible for the increase in Na+/H+ exchange function in sodium-depleted rats.


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Fig. 4.   Twenty-five micromolars HOE-694-sensitive (A) and HOE-694-insensitive (B) component of proton gradient-driven 22Na uptake. AMV isolated from proximal colon of normal (open bars) and sodium-depleted (solid bars) rats were preloaded with 150 mM K+-gluconate and 50 mM Mes-Tris (pH 5.5). Uptake was measured for 6 s by incubating the vesicles in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). Uptake in the presence of either 5 µM EIPA or 25 µM HOE-694 was also measured. Twenty-five micromolars HOE-694-sensitive component (A) or NHE2 isoform-specific fraction represents the difference between uptake in presence of 25 µM HOE-694 and that in its absence, and the remaining uptake or the HOE-694-insensitive component (B) is equivalent to NHE3 isoform activity. Both the HOE-694-sensitive and HOE-694-insensitive fraction are EIPA sensitive. EIPA-insensitive fractions represent 22Na uptake in the presence of EIPA and were subtracted from total Na+/H+ exchange activity. All media contained 25 µM valinomycin and 0.8% ethanol. * P < 0.05; **P < 0.01.

Western blot analyses. Although previous studies have established that NHE2 and NHE3 isoform-specific proteins are localized to the apical membrane of proximal colon of normal rats (5, 6, 8), only NHE3 isoform-specific protein expression was increased by aldosterone (7). Because the functional 22Na uptake studies (Fig. 4) revealed that both NHE2 and NHE3 activities were increased by sodium depletion, Western blot analyses were performed with apical membranes isolated from proximal colon of normal and sodium-depleted rats using a polyclonal antibody specific to NHE2 isoform (19) and a monoclonal antibody specific to NHE3 isoform (2). Figure 5A demonstrates that both NHE2 and NHE3 isoform-specific proteins were expressed in apical membranes of proximal colon of normal rats. The expression of both NHE2 and NHE3 isoform-specific proteins was substantially increased by approximately threefold and sevenfold, respectively (Fig. 5B), in apical membranes from proximal colon of sodium-depleted rats. In contrast, neither NHE2 nor NHE3 isoform-specific proteins were expressed in basolateral membranes of normal or experimental animals (data not shown).


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Fig. 5.   A: Western blot analysis of apical membranes of normal and sodium-depleted rat proximal colon. Blots were stained with NHE2 (1:2,000) and NHE3 (1:1,000) isoform-specific antibodies (2, 19) followed by horseradish peroxidase-conjugated secondary antibody, as described in METHODS. Size and position of protein markers in kilodaltons are shown at right. beta -Actin expression was not altered by sodium depletion (data not shown). B: relative abundances of NHE2 and NHE3 isoform protein in apical membrane of normal (open bars) and sodium-depleted (solid bars) rats were quantitated by densitometry from three different membrane preparations. * P < 0.05.

Northern blot analyses. Our observations establish that sodium depletion markedly increases both NHE2 and NHE3 activities and protein expression. Therefore, to determine whether these observations are due to transcriptional events, NHE isoform-specific mRNA abundance was examined by Northern blot analyses. The results shown in Fig. 6A demonstrate that NHE1, NHE2, and NHE3 cDNA probes hybridize with 4.8-, 4.4-, and 5.6-kb messages, respectively, in mRNA isolated from normal rat proximal colon and confirm previous studies of the presence of NHE isoform mRNA in rat colon (22, 33). Although all three NHE isoform-specific mRNAs are also expressed in proximal colon of sodium-depleted rats, only NHE2 and NHE3 mRNA abundances were substantially increased in the experimental animals (Fig. 6A). As shown in Fig. 6B, quantitation of NHE isoform-specific mRNA abundance using GAPDH as an internal control revealed that sodium depletion enhanced both NHE2 and NHE3 isoform-specific mRNA levels by 1.9- and 3.0-fold, respectively. In contrast, NHE1 isoform-specific mRNA level was not altered in sodium-depleted rats (Fig. 6B). These results suggest that sodium depletion stimulates electroneutral Na+ absorption by enhancing apical (i.e., NHE2 and NHE3), but not basolateral (i.e., NHE1), membrane-specific NHE isoform-specific mRNAs in rat proximal colon via pretranslational regulation.


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Fig. 6.   Expression of NHE isoform-specific mRNAs and their regulation by sodium depletion. A: mRNA purified from colonocytes of proximal colon of normal (left lane) and sodium-depleted (right lane) rats was analyzed by Northern blot hybridization using NHE isoform-specific (NHE1, NHE2, and NHE3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific cDNAs, as described in METHODS. Size and position of RNA markers in kilobases are shown at right. B: NHE isoform-specific mRNA abundances in normal (open bars) and sodium-depleted (solid bars) rats were quantitated by densitometry from 5 different mRNA preparations. Arbitrary units presented were normalized to GAPDH mRNA abundance. * P < 0.05.

Distal Colon

Previous studies established that the effect of sodium depletion and aldosterone1 on Na+ transport in the distal colon differs qualitatively from their effects in proximal colon (13, 15). Aldosterone inhibits both electroneutral Na+ absorption in intact tissue (18) and Na+/H+ exchange in AMV (26). As a result, the experiments in the distal colon examined the effects of sodium depletion on NHE isoforms by determining NHE isoform-specific 22Na uptake, protein, and message in normal and sodium depleted-animals.

22Na uptake studies. Previous studies of proton gradient-driven 22Na uptake have been performed in AMV from normal and sodium-depleted rats (26). Proton gradient-driven 22Na uptake was identified in AMV from distal colon of both groups but in the sodium-depleted animals was inhibited by low-dose amiloride (26), suggesting that proton gradient-driven 22Na uptake in the experimental animals represented proton diffusion-coupled electrogenic uptake (26). To confirm this possibility, proton gradient-driven 22Na uptake under nonvoltage clamped condition was assessed in AMV prepared from distal colon of normal and sodium-depleted rats. First, the effect of 1 µM EIPA on proton gradient-driven 22Na uptake was determined (Fig. 7A). Because 80% of proton gradient-driven 22Na uptake in AMV from normal animals was inhibited by 1 µM EIPA, Na+/H+ exchange is responsible for the major fraction of Na uptake across AMV in control rats. The results shown in Fig. 7A with benzamil, an amiloride analog that blocks Na+ channels but does not inhibit NHE activity, confirm our previous findings (26) that Na+ channels are not present in apical membrane of normal rat colonocytes. The effect of both EIPA and benzamil on proton gradient-driven 22Na uptake in AMV from sodium-depleted rats differs substantially from that seen in normal rats. Figure 7B demonstrates that proton gradient-driven 22Na uptake was inhibited by benzamil by 95% but is not inhibited by EIPA. These results indicate that Na+/H+ exchange is present in AMV from distal colon of normal but not of sodium-depleted rats, whereas in the sodium-depleted animals proton gradient-driven 22Na uptake occurs via apical membrane Na+ channels.


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Fig. 7.   Effect of inhibitors on proton gradient-driven 22Na uptake. AMV isolated from distal colon of normal (A) and sodium-depleted (B) rats were preloaded with 150 mM K+-gluconate and 50 mM Mes-Tris (pH 5.5). Uptake was measured by incubating the AMV in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). Uptake in medium with 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM Mes-Tris (pH 5.5) was also measured. Proton gradient-driven uptake presented was calculated by subtracting uptake obtained in medium with 50 mM Mes-Tris (pH 5.5) from that in medium with 50 mM HEPES-Tris (pH 7.5). Controls are presented as open bars. Uptake in the presence of either 1 µM of EIPA (solid bars), 1 µM HOE-694 (hatched bars), or 1 µM benzamil (gray bars) were also measured. Experimental groups with different letters are statistically significantly different at P < 0.01.

To determine the NHE isoform(s) responsible for Na+ uptake across apical and basolateral membrane of normal rats, the effect of 1 µM HOE-694 on proton gradient-driven 22Na uptake was also examined. Figure 7A presents results that are similar to those seen in proximal colon. Proton gradient-driven 22Na uptake in AMV was not inhibited by 1 µM HOE-694; in contrast, proton gradient-driven 22Na uptake in BLMV of both normal and sodium-depleted animals was effectively abolished (data not shown). Thus proton gradient-driven 22Na uptake in AMV from distal colon is not mediated by NHE1 isoform but by NHE2 and/or NHE3 isoforms. In contrast, the proton gradient-driven 22Na uptake in BLMV is regulated by NHE1 isoform.

Studies to distinguish NHE2 and NHE3 isoforms activities. To establish whether NHE2 and/or NHE3 isoform is responsible for Na+ movement across apical membrane, the effect of 25 µM HOE-694 on proton gradient-driven 22Na uptake in AMV from normal distal colon was also determined under voltage-clamped conditions. Similar to our earlier observation (26), voltage clamping did not affect proton gradient-driven 22Na uptake in AMV from normal rat, but substantially inhibited in AMV from the sodium-depleted animal (12.6 ± 1.3 vs. 0.0 ± 0.4 pmol · mg protein-1 · 6 s-1). Figure 8 demonstrates that 25% of proton gradient-driven 22Na uptake in AMV of normal rats was inhibited by 25 µM HOE-694, indicating that NHE2 isoform accounts for ~25% of basal apical membrane Na+/H+ exchange activity. In contrast, ~75% of total proton gradient-driven 22Na uptake is insensitive to 25 µM HOE-694 and represents NHE3 isoform. Sodium depletion virtually abolished NHE3 isoform component (Fig. 8B) while substantially reducing the NHE2 isoform component of proton gradient-driven 22Na uptake (Fig. 8A).


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Fig. 8.   Twenty-five micromolar HOE-694-sensitive (A) and HOE-694-insensitive (B) component of proton gradient-driven 22Na uptake. AMV isolated from proximal colon of normal (open bars) and sodium-depleted (solid bars) rats were preloaded with 150 mM K+-gluconate and 50 mM Mes-Tris (pH 5.5). Uptake was measured for 6 s by incubating the vesicles in medium containing 150 mM K+-gluconate, 0.1 mM Na+-gluconate, trace of 22Na, and 50 mM HEPES-Tris (pH 7.5). Uptake in the presence of either 5 µM EIPA or 25 µM HOE-694 was also measured. Twenty-five micromolar HOE-694-sensitive component (A) or NHE2 isoform-specific fraction represents the difference between uptake in presence of 25 µM HOE-694 and that in its absence, and the remaining uptake or the HOE-694-insensitive component (B) is equivalent to NHE3 isoform activity. Both the HOE-694-sensitive and HOE-694-insensitive fractions are EIPA sensitive. EIPA-insensitive fractions represent 22Na uptake in the presence of EIPA and were subtracted from total Na+/H+ exchange activity. All media contained 25 µM valinomycin and 0.8% ethanol. * P < 0.05; **P < 0.001.

Western blot analyses. Western blot analyses confirmed the presence of NHE2 and NHE3 isoform-specific proteins in apical membranes of distal colon in normal rats (Fig. 9A). These studies also established that sodium depletion decreased NHE2 isoform-specific protein expression by 33%, while NHE3 isoform-specific protein expression was decreased by 75% (Fig. 9B). These observations in sodium depletion parallel directly with the results of 22Na uptake studies.


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Fig. 9.   A: Western blot analysis of apical membranes of normal and sodium-depleted rat distal colon. Blots were stained with NHE2 (1:2,000) and NHE3 (1:1,000) isoform-specific antibodies (2, 19) followed by horseradish peroxidase-conjugated secondary antibody, as described in METHODS. Size and position of protein markers in kilodaltons are shown at right. beta -Actin expression was not altered by sodium depletion (data not shown). B: relative abundances of NHE2 and NHE3 isoform protein in apical membrane of normal (open bars) and sodium-depleted (solid bars) rats were quantitated by densitometry from 3 different membrane preparations. * P < 0.05; **P < 0.01.

Northern blot analyses. To determine whether the decrease in both NHE2 and NHE3 activities (Figs. 7 and 8) and protein expression (Fig. 9) by sodium depletion in distal colon represents transcriptional regulation, NHE isoform-specific mRNA abundance was examined by Northern blot analyses. Figure 10A presents NHE isoform-specific mRNA levels and reveals that NHE1, NHE2, and NHE3 isoform-specific mRNAs are present in distal colon of normal rats. Figure 10 also reveals that sodium depletion markedly reduced NHE2 isoform-specific mRNA levels and almost completely abolished NHE3 isoform-specific mRNA levels. Sodium depletion did not alter NHE1 isoform-specific mRNA levels. The results with NHE isoform-specific message parallel the observations that were obtained with HOE-694 inhibition of proton gradient-driven 22Na uptake and NHE isoform-specific protein. This result differs substantially from the changes in NHE2 and NHE3 isoform-specific mRNA abundance seen in proximal colon of sodium-depleted animals (Fig. 6). Sodium depletion upregulates NHE2 and NHE3 isoform-specific mRNA levels in proximal colon but downregulates NHE2 and NHE3 isoform-specific message levels in distal colon. The changes in NHE isoforms observed in both proximal and distal colon appear to represent pretranslational regulation of Na+/H+ exchange by dietary sodium depletion.


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Fig. 10.   Expression of NHE isoform-specific mRNAs and their regulation by sodium depletion. A: mRNA purified from colonocytes of distal colon of normal (left lane) and sodium-depleted (right lane) rats were analyzed by Northern blot hybridization using NHE isoforms and GAPDH-specific cDNAs, as described in METHODS. Size and position of RNA markers in kilobases are shown at right. B: NHE isoform-specific mRNA abundances in normal (open bars) and aldosterone-treated (solid bars) rats were quantitated by densitometry from 5 different mRNA preparations. Arbitrary units presented are normalized to GAPDH mRNA abundance. * P < 0.05; **P < 0.01.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

There are several significant differences in the characteristics and regulation of electroneutral NaCl absorption in proximal and distal colon (3, 4, 13, 18, 23, 24). First, electroneutral Na+ absorption in rat distal colon is Cl--dependent, and net Na+ absorption is approximately equivalent to net Cl- absorption (13). The demonstration of Na+/H+ exchange and Cl-/HCO-3 exchange in isolated AMV suggested that electroneutral NaCl absorption in distal colon occurs via the coupling of these two dual-ion exchanges by intracellular pH (23, 24). Second, in contrast in proximal colon, although electroneutral Na+ absorption is also Cl--dependent, net Cl- absorption is not present (13). Third, either sodium depletion or aldosterone inhibits both Na+ and Cl- absorption in distal colon when studied in vitro across isolated colonic mucosa under voltage clamp conditions, while inducing both net Na+ and Cl- absorption in proximal colon (13, 18). Recent studies in proximal colon indicated that aldosterone enhanced NHE3 isoform, but not NHE2 isoform, at a pretranslational level (7). These studies did not reveal any effects of aldosterone on NHE function in the distal colon.

The effect of glucocorticoids and aldosterone on Na+ and Cl- absorption differs strikingly in proximal colon (4). Although glucocorticoids and aldosterone both enhance electroneutral NaCl absorption in proximal colon of the rat, their effects are distinct and appear mediated by separate receptors. Aldosterone stimulates both electroneutral NaCl absorption that is inhibited by spironolactone, a mineralocorticoid receptor antagonist, and electrogenic K+ secretion (31). In contrast, RU-28362, a glucocorticoid receptor-specific agonist, stimulates spironolactone-insensitive NaCl absorption and does not affect K+ transport (31). These observations are best explained by separate and distinct corticosteroid receptors that regulate the effects of glucocorticoids and aldosterone on Na+/H+ exchange in the proximal colon.

These present studies demonstrate that sodium depletion upregulates both NHE2 and NHE3 isoform-specific mRNA, protein, and Na+/H+ exchange activity in proximal colon (Figs. 4-6). In contrast, prior studies revealed that dexamethasone increased NHE3, but not NHE2 isoform-specific mRNA and protein abundance (8). Similar observations with methylprednisolone, a glucocorticoid agonist, have recently been reported in rabbit ileum (35, 36). As a result, it would appear that although both aldosterone and glucocorticoids increase Na+/H+ exchange activity, their effects on NHE isoform-specific mRNAs are not identical. Aldosterone affects both NHE2 and NHE3 isoforms, whereas glucocorticoids regulate only the NHE3 isoform.

The present results indicate that the NHE3 isoform, which represents 89% of the EIPA-sensitive Na+/H+ exchange in AMV (Fig. 4), is the primary NHE isoform responsible for electroneutral Na+ absorption in the proximal colon of normal rats. Because sodium depletion increased NHE2 isoform-specific Na+/H+ exchange activity considerably more than NHE3 isoform-specific Na+/H+ exchange function, we suspect that NHE2 isoform, but not NHE3 isoform, is primarily responsible for the stimulation of electroneutral Na+ absorption by sodium depletion in proximal colon. This possibility is further supported by the recent observation that aldosterone induced Na+ absorption in avian colon by selectively stimulating the NHE2 isoform (11). Our results, however, differ from those of Cho et al (7), who demonstrated that NHE3 isoform, but not NHE2 isoform, message abundance was increased by aldosterone in proximal colon of rat. This discrepancy may be due to different experimental maneuvers used in these two studies. Cho et al. (7) produced the hyperaldosterone state by intraperitoneal injection of aldosterone for 3 days, while in the present study increased plasma aldosterone levels were the result of 7 days of dietary sodium depletion. Although previous studies have established that dietary sodium depletion for 7 days and continuous subcutaneous infusion of aldosterone via minipumps for 7 days produce identical changes of colonic Na+ and Cl- transport (15),1 it should be noted that intraperitoneal administration of aldosterone for 3 days resulted in serum aldosterone levels that are 24% less than those seen both in dietary sodium depletion for 7 days and in animals that had received continuous subcutaneous administration of aldosterone for 3 days (7); it is not known whether enhancement of NHE2 isoform-specific abundance requires elevated aldosterone levels for more than 3 days.

Similar to observations in proximal colon (Fig. 4), both NHE2- and NHE3-specific Na+/H+ exchange activities are expressed in AMV of the distal colon of normal rats (Fig. 8). NHE3 activity represents almost 87%, while the remaining EIPA-sensitive Na+/H+ exchange activity represents NHE2 isoform in AMV of normal distal colon. In contrast to proximal colon, sodium depletion inhibited both NHE2 and NHE3 isoform-specific mRNA abundance, protein expression, and Na+/H+ exchange activities but not NHE1 isoform-specific mRNA abundance and Na+/H+ exchange activity (Figs. 8-10). The inhibition of NHE2 isoform protein by sodium depletion was substantially less than that of Na+/H+ exchange activity and would be compatible with a decrease in turnover rate of NHE2 isoform protein. Although Cho et al. (7) demonstrated enhanced NHE3 message by aldosterone in proximal colon, they also reported that aldosterone did not have any effect on NHE isoform message and protein abundance in the distal colon. It is likely that their failure to observe changes in NHE isoform message and protein may also be related to the different methods used to enhance serum aldosterone levels and the duration during which aldosterone levels were elevated. Although dietary sodium depletion and subcutaneous administration of aldosterone for 7 days resulted in identical inhibition of both Na+/H+ exchange in AMV and electroneutral NaCl absorption in the distal colon, electroneutral NaCl absorption was not inhibited by 3 days of subcutaneous administration of aldosterone (18). Thus it is likely that the differences in observations between these present results and those of Cho et al. (7) are likely due to the shorter (3 days) period that aldosterone was administered and/or the lower serum aldosterone levels achieved by the intraperitoneal route of its administration.

The present data indicate that NHE isoforms that are localized in colonic apical membrane are regulated by aldosterone at a pretranslational level because these isoform-specific mRNA abundances, protein expressions, and Na+/H+ exchange activities were altered in parallel. It is highly likely that the effect of sodium depletion to stimulate NHE3 isoform-specific message, protein, and Na+/H+ exchange activities in rat proximal colon (Figs. 4-6; Ref. 7) is not a consequence of aldosterone's activation of the glucocorticoid receptor (31). Because the stimulation of electroneutral Na+ absorption by aldosterone in the proximal colon is inhibited by spironolactone (31), a mineralocorticoid-receptor antagonist, we conclude that both NHE2 and NHE3 isoforms are regulated at a pretranslational level by aldosterone following its interaction with the mineralocorticoid receptor in rat colon. However, the mechanism of regulation of apical membrane NHE isoforms by aldosterone differs in a tissue-specific pattern, because NHE2 and NHE3 mRNA abundance, protein expression, and Na+/H+ exchange activities are upregulated in proximal (Figs. 4-6), but downregulated in distal, colon (Figs. 8-10). More than one explanation could account for this tissue-specific differential regulation of NHE mRNA and Na+/H+ exchange activities. First, identical NHE isoforms are present in both proximal and distal colon but are under the control of different promoters. Therefore, it is likely that these differential effects of aldosterone in the proximal and distal colon are the result of the tissue-specific localization of different aldosterone-responsive promoters in proximal and distal colon. Second, it is also possible that apical membrane NHE isoforms expressed in proximal and distal colon represent highly homologous but not identical isoforms similar to that recently shown for anion exchange isoforms with NH2-terminal variations (34). However, this hypothesis will require further identification and sequencing for substantiation.

In summary, both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities are present in AMV, while NHE1 isoform is present in BLMV of proximal and distal colon of normal rats. Sodium depletion inhibits electroneutral Na+ absorption by reducing both NHE2 and NHE3 isoforms in distal colon while it enhances Na+ absorption in proximal colon by stimulating both NHE2 and NHE3 isoforms. In contrast, NHE1 isoform in proximal or distal colon was not altered by sodium depletion. Regulation of apical membrane-specific NHE isoforms by dietary sodium depletion occurs at a pretranslational level and is both tissue specific and isoform specific.


    ACKNOWLEDGEMENTS

Prof. R. Gregor generously provided HOE-694. We thank Dr. Sarah Kolla for advice about cDNA probe preparation and Andrea Mann for performing the Western blot analyses.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-14669-25.

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. §1734 solely to indicate this fact.

1 Previous studies demonstrated identical changes in Na+ transport in dietary Na+-depleted animals and rats given aldosterone subcutaneously via osmotic minipumps (15). Serum aldosterone levels were equivalent in these two experimental groups (15). As a result, the experimental groups used in this present study are referred to as either aldosterone-treated or Na+-depleted rats.

2 HOE-694 inhibits 22Na uptake in PS120 cells that had been individually transfected with NHE1, NHE2, and NHE3 isoform cDNAs with half-maximal inhibitory concentration of 0.16 mM, 5 µM, and 650 µM, respectively (9). As a result of these previous observations, the NHE1 isoform in these present studies of 22Na uptake by colonic AMV is defined as proton gradient-driven 22Na uptake that is inhibited by 1 µM HOE-694. NHE2 isoform is defined as proton gradient-driven 22Na uptake inhibited by 25 µM HOE-694, and proton gradient-driven 22Na uptake that is insensitive to 25 µM HOE-694 represents NHE3 isoform activity.

Address for reprint requests: V. M. Rajendran, Dept. of Internal Medicine, Yale Univ. School of Medicine, Box 208019, 333 Cedar St., New Haven, Connecticut 06520.

Received 31 August 1998; accepted in final form 26 October 1998.


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Discussion
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