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MUCOSAL BIOLOGY

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na⁺-dependent acid extrusion mechanism is upregulated in NHE2-null mice

Departments of ¹Molecular and Cellular Physiology and ³Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio; and ²Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 22 July 2005 ; accepted in final form 9 May 2006

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
The mechanism of apical Na⁺-dependent H⁺ extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH_i) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na⁺ stimulated similar rates of pH_i recovery in cells at the base of distal colonic crypts of wild-type or Na⁺/H⁺ exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 µM HOE694 (NHE2 inhibitor) blocked 68–75% of the pH_i recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 µM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 µM), inhibited pH_i recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na⁺ leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na⁺-dependent pH_i recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH_i recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na⁺-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.

Slc9a2; Slc9a3; BCECF; SNARF-5F; intracellular pH; laser scanning confocal microscopy; colon; Na⁺/H⁺ exchanger

The role of NHE3 in intestinal and/or colonic Na⁺ absorption has been clearly demonstrated by the occurrence of diarrhea in NHE3-null mice (25). The absence of a diarrhea phenotype or any apparent Na⁺-absorptive defect in NHE2-null mice was taken as evidence that NHE2 does not play an important role in Na⁺ absorption (13, 24). In addition, diarrhea in NHE3-null mice was not further aggravated in NHE2/NHE3 double-knockout mice (19), as would have been expected if NHE2 provided a major absorptive capacity. However, rat colonic NHE2 and NHE3 are similarly increased by Na⁺ depletion (17). On the basis of inhibitor sensitivity, NHE2 but not NHE3 appeared to be responsible for the enhanced Na⁺ absorption in response to a low-Na⁺ diet in the chicken colon (11). Pharmacological evidence also suggests that NHE2 rather than NHE3 may be important for basal Na⁺ absorption in the rat colon (8). Thus, the function of NHE2 in the colon remains uncertain.

NHE2 and NHE3 mRNA and protein are expressed at high levels in the colon (12, 16, 20), but they have different spatial distributions. Whereas NHE3 mRNA and protein seem to be present almost exclusively in surface cells (2, 7, 9, 12, 16), NHE2 mRNA and protein do not only occur in surface cells but extend downward into the crypt (2, 7) and possibly occur also at the crypt base (2, 7, 9, 12, 16). In mouse colonocytes, robust apical Na⁺/H⁺ exchange has been detected near the base of colonic crypts (9). This activity is stimulated by short-chain fatty acids in the luminal fluid and could play a role in the absorption of these acids (9). Pharmacological studies of isolated crypt cells or of crypts in intact epithelial sheets have suggested that NHE2 best matches the identified transport activity in both proximal and distal colonic crypts of wild-type mice (2, 9). This view is supported by immunostaining showing NHE2-like epitopes near the crypt base (7, 9). However, these data must be interpreted with caution. In isolated colonic crypt cells, Na⁺/H⁺ exchange activity was similar between wild-type and NHE2-null mice (2, 13), indicating that the loss of NHE2 did not significantly impair total Na⁺/H⁺ exchange. It was suggested that the observed twofold increase in NHE3 mRNA may be part of a compensatory response in NHE2-null mice that explains why eliminating NHE2 did not affect net Na⁺ transport (2). However, in this system, it was not possible to determine whether the observed transport activity was located at the apical membrane or whether NHE3 was upregulated in the crypt cells that normally express NHE2.

The purpose of the present study was to determine whether NHE2 mediates apical Na⁺/H⁺ exchange near the base of mouse colonic crypts. To explore this possibility, we employed a system in which distal colonic epithelia from wild-type mice and NHE knockout mice (as negative controls) are mounted in a chamber that allows independent superfusion of both mucosal and serosal solutions while simultaneously imaging pH_i in colonic epithelial cells by laser scanning confocal microscopy. These experiments, along with immunofluorescence cytochemistry, allowed us to unambiguously evaluate 1) the expression and activity of NHE2 and NHE3 in colonic crypts and at the colonic surface; 2) the effects of NHE2 disruption on both apical Na⁺/H⁺ exchange and the distribution of NHE2- and NHE3-immunoreactive epitopes; and 3) the possibility that either NHE3 or paracellular Na⁺ leakage, possibly in combination with basolateral NHE1 activity, mediates the pH_i response to luminal Na⁺ stimulation near the crypt base. The results provide the first conclusive evidence of apical NHE2 activity in the colon, show that NHE2 is the major apical NHE in colonic crypts, and demonstrate that NHE3 is not the 5-(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive compensatory mechanism that is upregulated in the apical membrane of NHE2-null crypt colonocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Animals. We used mutant mice with targeted disruption (knockout) of genes encoding NHE1 (Slc9a1), NHE2 (Slc9a2), and NHE3 (Slc9a3). At 1–3 wk of age, all offspring were genotyped using specific primers for murine NHE1, NHE2, and NHE3 and the disrupted alleles (4, 24, 25). Experiments were performed using tissue obtained from mice at 1–2 mo of age for NHE1 and NHE3 knockout colonies and at 2–4 mo of age for the NHE2 knockout colony. All experimental protocols were approved by the University of Cincinnati Animal Care and Use Committee and complied with National Institutes of Health guidelines.

Colon preparation for perfusion. After being anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ), animals were cervically dislocated, and muscle-stripped distal colonic mucosal sheets prepared. Isolated tissue was kept in DMEM (GIBCO-BRL, Grand Island, NY) on ice and used within 4 h, as previously described (10). For studies of crypts, tissue was loaded with 3 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM (Molecular Probes, Eugene, OR) at room temperature for 20 min in Na⁺ medium containing (in mM) 130 NaCl, 5 KCl, 1 MgSO₄, 2 CaCl₂, 1 (Na)PO₄, 20 HEPES, 25 mannose, and 1 probenecid; pH 7.4. For studies of surface cells, tissue was loaded with 5 µM SNARF-5F 5-(and-6)-carboxylic acid-AM acetate (SNARF-5F-AM; Molecular Probes) under otherwise identical conditions. This switch in dyes was necessary because of the high autofluorescence and high dye leakage from surface cells when BCECF was used (data not shown). For experiments, a mucosal sheet was mounted in a microscope chamber on a Zeiss LSM510 confocal microscope (Zeiss, Jena, Germany) such that the luminal and serosal surfaces could be superfused independently. The chamber was mounted with the luminal (for surface cell study) or serosal (for crypt base study) surface of the sheet facing the objective lens (Zeiss C-Apo x40 or C-Apo x10, water immersion). The chamber for studies of crypts was as described previously (10) but had to be adapted to study surface cells because of issues of mucus obscuring the images and tissue instability. For surface cell studies, tissue was mounted between two concentric rings (less stressing of the surface that would lead to mucin secretion and greater tissue stability for imaging) before being mounted in the perfusion chamber.

Perfusate solutions. Perfusate solutions were based on the Na⁺ medium. In Na⁺-free solution, tetramethylammonium chloride (TMA-Cl) replaced all NaCl (mol:mol), whereas in the ammonium medium, 25 mM NH₄Cl replaced equimolar NaCl or TMA-Cl. The NHE blocker EIPA was from Sigma (Natick, MA). HOE694 and S-1611 (generous gifts from Dr. H. J. Lang, Aventis Pharma Deutschland, Frankfurt/Main, Germany) were used to inhibit NHE2 and NHE3, respectively. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; Research Organics, Cleveland, OH) was used to test for the presence of the Na⁺-HCO₃^– cotransporter. All solutions were prepared directly before use and adjusted to pH 7.4. In some cases, 100 µM lucifer yellow (LY; CH lithium salt, Molecular Probes) was added to superfusates.

Confocal microscopy measurement of pH_i. Confocal microscopy was carried out as described previously (10) with modifications. Briefly, for studying colonic crypts, tissue was loaded with BCECF, and ratiometric measurements of dye fluorescence (alternating 488/458-nm excitation, >505-nm emission) were performed to assess the pH_i while tissue was focused near the crypt base. The plane of focus was selected to be directly adjacent to the base of crypts such that the crypt lumen was just visible and crypt epithelial cells within the image could be visualized along their apical-to-basal pole (9). We used 60% of 458-nm laser power and 3% of 488-nm laser power for BCECF, and each image was collected in 3 s. No photobleaching was observed, even after imaging of the same region for 120 min (data not shown). At the end of the time-course experiments measuring pH_i, imaging parameters were switched to measure a time course of LY fluorescence in the same region (458-nm excitation, 620- to 680-nm emission), avoiding optical contamination by BCECF. Membrane-impermeant LY (100 µM) was added sequentially to luminal and then luminal plus serosal perfusates to quantify transepithelial LY leakage. The routine measurement of transepithelial leakage was made when LY was only added to the luminal perfusate. In this condition, the average intensity of LY in the lumen was divided by the intensity of LY in the lamina propria adjacent to the crypt (avoiding areas where LY had bound to connective tissue). This LY ratio provided a measure for the degree of egress of LY from the lumen to serosa (e.g., a LY ratio of 1 means complete equilibration). To measure colonic surface epithelial cells loaded with SNARF-5F, fluorescence was excited at 543-nm excitation (with 15% of the laser power) and 550- to 600-/620- to 680-nm emission wavelengths measured simultaneously. Images were analyzed using Metamorph software (Universal Imaging, Downingtown, PA).

Immunocytochemistry. Chemical tissue fixation, sectioning, immunostaining, and confocal imaging of NHE2 and NHE3 epitopes were performed exactly as described previously (10), but the antiserum to NHE2 was used in 1:100 dilution and that to NHE3 in 1:900 dilution. The polyclonal antisera against NHE2 (rat NHE2 amino acids 676–813) and NHE3 (rat NHE3 amino acids 528–648) had been produced in rabbits (kindly provided by Drs. E. B. Chang and M. Musch, University of Chicago, Chicago, IL). Preimmune sera (by Drs. E. B. Chang and M. Musch) were used in 1:100 dilution. Secondary antiserum was used in 1:400 dilution (Alexa 488-labeled goat anti-rabbit IgG, Molecular Probes).

Statistics. Data between two groups were compared using the two-tailed paired or unpaired Student's t-test. Differences were considered to be statistically significant at = 5%. Data are expressed as means ± SE. Data statistics were performed using GraphPad Prism (GraphPad Software, San Diego, CA).

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Apical Na⁺/H⁺ exchange activity near the base of NHE2-null mice distal colonic crypts. Previously, inhibitor profiles suggested that NHE2 is the most likely candidate for an apical NHE in colonocytes near the base of colonic crypts (9). These studies used confocal microscopy to report the pH_i from intact epithelial sheets of the normal mouse distal colon. Previous use of NHE2 knockout animals to assess NHE2 functions in colonic crypts had been restricted to a study of isolated crypt colonocytes of uncertain location along the crypt-to-surface axis from the proximal colon (2). We therefore used confocal microscopy to first compare the response to luminal Na⁺ in NHE2-null versus wild-type mice. An NH₄Cl prepulse was used to acidify colonocytes, and a transition from Na⁺-free (TMA containing) medium to 140 mM Na⁺ medium in either the luminal or serosal perfusate was used to activate apical or basolateral Na⁺/H⁺ exchange, respectively, leading to the recovery of resting pH_i. A second round of acidification and Na⁺ return was carried out, confirming that pH_i recovery was robust and reproducible and that any potential photodamage to tissue was not sufficient to alter cell function in this setting.

As shown, the colonocytes near the crypt base from wild-type (Fig. 1A) and NHE2-null mice (Fig. 1B) manifested a qualitatively and quantitatively similar pH_i recovery in response to luminal Na⁺ addition. There were no significant differences in the initial rates of pH_i recovery (pH_i/min) from the nadir pH_i either between genotypes or between the first and second round of pH_i recovery (wild-type mice: first round, 0.075 ± 0.011, n = 15 crypts from 2 animals; second round, 0.067 ± 0.007, n = 15 crypts; NHE2-null mice: first round, 0.077 ± 0.006, n = 16 crypts from 2 animals; second round, 0.077 ± 0.015, n = 16 crypts). The nadir pH_i after acidification was not different between wild-type mice (6.06 ± 0.04, n = 37 crypts from 5 animals) and NHE2-null mice (6.11 ± 0.03, n = 37 crypts from 5 animals). Similarly, the resting pH_i was not different between wild-type mice (6.94 ± 0.03, n = 37 crypts) and NHE2-null mice (7.11 ± 0.05, n = 37 crypts). Therefore, surprisingly, no difference between wild-type and NHE2-null mice was detected at this level.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Time course traces of intracellular pH (pHi) changes in crypts of intact distal colonic epithelial sheets. Tissues from wild-type mice (A; n = 15 crypts from 2 animals) and Na+/H+ exchanger isoform 2 (NHE2)-null mice (B; n = 16 crypts from 2 animals) were subjected to two successive rounds of acidification via a NH4+ prepulse and Na+ addition into the luminal (L) or serosal (S) compartments. After a sheet had been loaded with the pH-sensitive dye BCECF, cells were imaged near the base of the crypts with confocal microscopy. Na, Na+ medium; NH4, NH4Cl; TMA, tetramethylammonium in Na+-free medium. Data points are means ± SE from 2 representative experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2. pHi sensitivity of Na+ transport in wild-type and NHE2-null mice. A: scatter plot of correlation between the degree of NH4+-induced pHi acidification (acidified pHi) and the rate of apical pHi recovery (pHi/min) in wild-type mice (R2 = 0.41, n = 37 crypts from 5 animals) and NHE2-null mice (R2 = 0.11, n = 37 crypts from 5 animals); note the lack of any apparent difference between genotypes. B: same data as in A were subdivided into two smaller pHi ranges and mean ± SE rates were plotted. Differences between genotypes were not statistically significant (P > 0.05).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effects of specific NHE inhibitors on cells near the crypt in wild-type and NHE2-null mice. Tissues from wild-type and NHE2-null mice were treated as in Fig. 1, and either HOE694 or S-1611 was added to the luminal compartment after the second acidification via a NH4+ prepulse. Luminal 20 µM HOE694 (NHE2 inhibitor) inhibited the rate of colonocyte apical pHi recovery (pHi/min) in response to luminal Na+ addition in wild-type mice. However, neither HOE694 nor 20 µM S1611 (NHE3 inhibitor) inhibited the apical Na+ recovery rate in NHE2-null mice. Data are presented as means ± SE; n indicates the numbers of crypts in each group with data from 7 animals. *P < 0.05 vs. control (no inhibitor added) in the same genotype.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Effects of the general NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) in wild-type and NHE2-null mice. Experiments were performed as in Fig. 1, and results were analyzed as in Fig. 3. With the use of tissues from wild-type and NHE2-null mice, EIPA (20 µM) was added to the luminal compartment after the second NH4+ prepulse acidification. EIPA inhibited the rate of colonocyte apical pHi recovery (pHi/min) in response to luminal Na+ in both genotypes. ***P < 0.001 vs. control in the same genotype.

These results show that in response to luminal Na⁺, NHE2-null mice utilize a different mechanism to recover the pH_i rate than wild-type mice and that this mechanism is sensitive to EIPA but unlikely to be NHE3- or SITS-sensitive HCO₃^– transporter.

Apical Na⁺/H⁺ exchange activity in surface epithelial cells. Colonic surface epithelial cells, in contrast to crypt cells, have been reported to contain both NHE2 and NHE3 mRNA and protein (2, 6, 7, 9, 12, 16). Therefore, we used measurements of pH_i as a means to compare epithelial function in surface cells versus crypt cells of wild-type mice. Figure 5A shows that the colonic surface could be loaded with the pH sensor SNARF-5F. As shown in Fig. 5B, both 20 µM HOE694 and 20 µM S1611 caused a significant inhibition of the Na⁺-dependent recovery from an acid load in surface cells (33% or 57% inhibition of transport, respectively). In addition to supporting the presence of both apical NHE2 and NHE3 function in colonic surface cells, the results confirm the efficacy of S-1611 as an inhibitor in our experimental preparation and demonstrate striking functional differences between cells at the surface versus cells at the crypt base.

View larger version (55K): [in this window] [in a new window]   Fig. 5. Measurements of pHi in colonic surface epithelial cells and cellular responses to specific NHE inhibitors in wild-type mice. A: confocal images of the colonic surface during superfusion and after being loaded with 5 µM SNARF-5F [left, 550- to 600-nm emission (Em), middle, 620- to 680-nm emission; right, transmitted light image]. B: experiments were performed as in Fig. 1, and results were analyzed as in Fig 3. With the use of tissues from wild-type mice, S-1611 or HO694 (both 20 µM) was added to the luminal compartment after the second NH4+ prepulse acidification. Initial rates of pHi recovery in the presence of inhibitor were normalized to the initial rate of transport (pHi/min) observed in the absence of inhibitor from the same crypt (control = 100%). Results are presented as means ± SE for n = 5 tissues from 4 animals. *P < 0.05 vs. control; **P < 0.01 vs. control. Scale bars = 20 µm.

We next used the LY ratio to test whether enhanced transepithelial leakage of luminally added Na⁺ would stimulate basolateral Na⁺/H⁺ exchange activity more in NHE2-null mice than in wild-type mice. This required us to run a pH_i experiment and then afterward evaluate individual crypt LY ratios for the same crypts, so that rates of transport could be directly correlated. Figure 6 demonstrates that LY fluorescence could be measured completely separately from BCECF fluorescence. Figure 6A shows colonic crypts loaded with BCECF, whereas Fig. 6B reveals the same crypts after LY had been added to perfusates.

View larger version (83K): [in this window] [in a new window]   Fig. 6. Paired measures of pHi and paracellular leakiness in individual colonic crypts. A: pHi determined by ratio imaging using 3 µM BCECF [left, 488-nm excitation (Ex); middle, 458-nm Ex]. Right, intracellular BCECF ratio (488-to-458 nm) in pseudocolor, with the corresponding pHi scale indicated qualitatively by the color bar. B: at the end of each experiment, 100 µM of the fluorescent, membrane-impermeant lucifer yellow (LY; 458-nm Ex and 620- to 680-nm Em) was added to luminal and then to luminal + serosal perfusates to quantify transepithelial LY leakage. As shown, LY could be imaged without optical contamination by BCECF. In the presence of only luminal LY, the LY ratio was assessed as the intensity of LY in the lumen divided by the intensity of LY in the lamina propria adjacent to the crypt (S), providing a measure of LY egress from the lumen to serosa. *Cross-sectioned lumen near the crypt base. Scale bars = 20 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Correlation of paracellular leakiness versus the pHi response to luminal Na+. No correlation was apparent between the LY ratio (lumen/serosa) and apical pHi recovery rate (pHi/min) for either wild-type mice (R2 = 0.07, n = 27 crypts from 5 animals) or NHE2-null mice (R2 = 0.11, n = 33 crypts from 5 animals).

View larger version (12K): [in this window] [in a new window]   Fig. 8. Time course of pHi changes in colonic crypt cells of NHE1-null mice. Experiments were performed as in Fig. 1. A: luminal Na+ activated pHi recovery, but subsequent serosal Na+ did not accelerate the rise in pHi (n = 10 crypts from 2 NHE1-null mice) as observed in wild-type mice (see Fig. 1). B: 20 µM HOE694 strongly inhibited the rate of Na+-induced apical pHi recovery in NHE1-null mice (n = 21 crypts from 4 animals) compared with wild-type mice (n = 33 from 6 animals), consistent with the presence of NHE2 on the apical membrane. *P < 0.05 vs. control.

View larger version (68K): [in this window] [in a new window]   Fig. 9. Fidelity of immunostaining with NHE2 antiserum. Dual staining for NHE2 immunoreactivity (green) and nuclear staining with propidium iodide (red) is shown. A: NHE2 immunoreactivity in the apical brush border of colonocytes and near the base of colonic crypts in a wild-type mouse. B–D: no NHE2 immunoreactivity was seen in NHE2-null mice (B) or after the primary antiserum was replaced by preimmune serum (C and D). Crypts in the epithelium are viewed longitudinally. *Crypt lumen. Arrows point to surface epithelium. Images are representative of results from 3 pairs of animals. Scale bars = 20 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 10. Immunofluorescence localization of NHE3 in the mouse distal colon (surface-to-base view). A: NHE3 immunoreactivity (green) was at the brush border of surface colonocytes and in the upper fifth part of the crypts of wild-type mice. However, NHE2-null mice showed intense NHE3 immunoreactivity in the brush border of surface cells and in cells of the upper third part of the crypts (C). E: cells in the NHE3-null colon did not show any NHE3 immunoreactivity. B, D, and F: control stainings using preimmune instead of primary antiserum. Nuclei were stained with propidium iodide (red). *Crypt lumen. Arrows point to surface epithelium. Images are representative of 3 animals. Scale bars = 20 µm.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

Transepithelial Na⁺ leakage does not contribute to pH_i recovery in wild-type or NHE2-null mice. Previously, we identified dye leakage between the luminal and serosal compartment in our preparation of mouse colonic sheets (9). This raised the possibility that transepithelial leakage of Na⁺ from the mucosal to serosal solution activates basolateral NHE1 activity to become part of the colonocyte pH_i recovery response to luminal Na⁺ addition. Obviously, such a situation would confound reliable assessment of the activity of apical Na⁺/H⁺ exchange. This possibility might be tested by pharmacologically inhibiting NHE1 activity, but in our hands, none of the available blockers fully inhibited NHE1 in colonic epithelium without partially affecting other NHE isoforms as well (Y. Guan, unpublished observations). Here, we tested the influence of transepithelial Na⁺ leakage using NHE1-null mice and LY as a marker for paracellular leakage. We conclude that basolateral NHE1 activity is not activated by transepithelial leakage of luminally applied Na⁺, for the following three reasons. First, by checking the LY ratio between the luminal and serosal compartment before the experiment, we were able to discard preparations with intercompartmental leakage due to improperly mounted colonic sheets in the superfusion chamber. Second, assuming that a low LY ratio indicates strong paracellular leakage of LY, and presumably also of Na⁺, one would expect to find that low LY ratios would be negatively correlated to high, Na⁺-stimulated NHE1 activity and, hence, to a high pH_i recovery rate. However, we did not observe a correlation between the LY ratio and pH_i recovery rate. This indicates that paracellular leakage, although measurable, does not flux enough Na⁺ into the basolateral spaces to influence the response to luminal Na⁺. Finally, the use of NHE1-null mice enabled us to completely eliminate basolateral NHE activity. Because the mutants showed a normal HOE694-sensitive pH_i recovery rate upon luminal Na⁺ addition and no substantial Na⁺/H⁺ exchange during basolateral Na⁺ perfusion, an apical Na⁺-dependent acid extrusion mechanism must be present.

Importantly, we found that in the absence of NHE2, another apical Na⁺-dependent acid extrusion mechanism was upregulated. Using NHE2-null mice, Bachmann et al. (2) concluded that mutating the NHE2 gene did not diminish total Na⁺/H⁺ exchange activity in cells isolated from the mouse proximal colon. The present study extends this conclusion to the distal colon, because it shows a robust and reproducible pH_i recovery in colonocytes near the base of the distal colonic crypt, with a rate that did not differ between wild-type and NHE2-null mice. On the basis of RT-PCR, Bachmann et al. (2) found that isolated cells from colonic crypts have an increased expression of NHE3 mRNA in NHE2-null mice and suggested this as the compensatory mechanism sustaining transport activity (2). We extend this observation to the protein level, revealing by immunocytochemistry that NHE3 protein in NHE2-null mice extends deeper into crypts. However, no NHE3 protein was detected at the base of the crypt, and the NHE3 blocker S-1611 was ineffective as an inhibitor of pH_i recovery in response to luminal Na⁺ in the crypts of NHE2-null mice (although it was an effective inhibitor of transport in surface cells). Based on these observations, we conclude that NHE3 is unlikely to be the apical Na⁺-dependent acid extrusion mechanism that acts in the crypts of NHE2-null mice. Because EIPA, which inhibits most known NHE isoforms (20), blocks >50% of the apical pH_i recovery in NHE2-null mice, one candidate for this apical extrusion mechanism is another NHE isoform that is neither NHE3 nor NHE1. Because EIPA may also inadvertently affect non-NHE transporters, the results might also be explained by the action of an apical Na⁺-HCO₃^– cotransporter (23). However, the lack of effect of the HCO₃^– transport inhibitor SITS (5, 22) on pH_i recovery in the crypts of NHE2-null mice does not support this possibility. Alternatively, a combination of apical transporters that are indirectly linked (e.g., via pH or membrane potential changes) to evoke Na⁺-dependent net acid extrusion may play a role, because the significantly smaller effect of EIPA in NHE2-null mice suggests that multiple compensatory mechanisms, both EIPA sensitive and insensitive, may be involved. Irregardless, EIPA will be a useful tool for further exploration of the nature of at least one component of this upregulated transport.

NHE2-null mice do not show diarrhea, leading investigators to previously conclude that NHE2 does not play an important role in the absorption of Na⁺ and water in the colon of the normal mouse. However, previous data as well as our present observations indicate that lack of NHE2 leads to a compensatory response to replace the missing functionality of NHE2 in crypts (2). Because we have identified this compensatory response as apical transport activity, the possibility remains that NHE2 mediates at least a fraction of Na⁺ absorption in normal animals and that this role is replaced by an alternative mechanism in NHE2-null mice. It must be noted that, although crypts have been shown to mediate Na⁺-dependent fluid absorption (15, 26), at present there is no direct evidence that colonic crypts absorb Na⁺. However, the presence of a compensatory response to functionally replace an absent NHE2 provides compelling evidence that NHE2 serves a vital function in the crypts. Identifying the constellation of Na⁺-uptake mechanisms in the mouse colonic crypt, their relevance to net Na⁺ absorption, and the identity of the compensatory mechanism(s) caused by the absence of NHE2 are important goals of future research.

	GRANTS

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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-42457 (to M. H. Montrose) and RO1-DK-50594 (to G. E. Shull).

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. H. Montrose, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, Medical Sciences Bldg., Cincinnati, OH 45267-0576 (e-mail: mhm{at}uc.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.

	REFERENCES

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1. Aronson P. Identifying secondary active solute transport in epithelia. Am J Physiol Renal Fluid Electrolyte Physiol 240: F1–F11, 1981.[Abstract/Free Full Text]
2. Bachmann O, Riederer B, Rossmann H, Groos S, Schultheis PJ, Shull GE, Gregor M, Manns MP, and Seidler U. The Na⁺/H⁺ exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation. Am J Physiol Gastrointest Liver Physiol 287: G125–G133, 2004.[Abstract/Free Full Text]
3. Bailey MA, Giebisch G, Abbiati T, Aronson PS, Gawenis LR, Shull GE, and Wang T. NHE2-mediated bicarbonate reabsorption in the distal tubule of NHE3 null mice. J Physiol 561: 765–775, 2004.[Abstract/Free Full Text]
4. Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, Shull GE, and Scott WJ. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. Am J Physiol Cell Physiol 276: C788–C795, 1999.[Abstract/Free Full Text]
5. Biagi BA. Effects of the anion transport inhibitor, SITS, on the proximal straight tubule of the rabbit perfused in vitro. J Membr Biol 88: 25–31, 1985.[CrossRef][ISI][Medline]
6. Bookstein C, DePaoli AM, Xie Y, Niu P, Musch MW, Rao MC, and Chang EB. Na⁺/H⁺ exchangers, NHE-1 and NHE-3, of rat intestine. Expression and localization. J Clin Invest 93: 106–113, 1994.[ISI][Medline]
7. Bookstein C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao MC, and Chang EB. Tissue distribution of Na⁺/H⁺ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell Physiol 273: C1496–C1505, 1997.[Abstract/Free Full Text]
8. Cermak R, Lawnitzak C, and Scharrer E. Influence of ammonia on sodium absorption in rat proximal colon. Pflügers Arch 440: 619–626, 2000.[ISI][Medline]
9. Chu J, Chu S, and Montrose MH. Apical Na⁺/H⁺ exchange near the base of mouse colonic crypts. Am J Physiol Cell Physiol 283: C358–C372, 2002.[Abstract/Free Full Text]
10. Chu S, Brownell WE, and Montrose MH. Quantitative confocal imaging along the crypt-to-surface axis of colonic crypts. Am J Physiol Cell Physiol 269: C1557–C1564, 1995.[Abstract/Free Full Text]
11. Donowitz M, De La Horra C, Calonge ML, Wood IS, Dyer J, Gribble SM, De Medina FS, Tse CM, Shirazi-Beechey SP, and Ilundain AA. In birds, NHE2 is major brush-border Na⁺/H⁺ exchanger in colon and is increased by a low-NaCl diet. Am J Physiol Regul Integr Comp Physiol 274: R1659–R1669, 1998.[Abstract/Free Full Text]
12. Dudeja PK, Rao DD, Syed I, Joshi V, Dahdal RY, Gardner C, Risk MC, Schmidt L, Bavishi D, Kim KE, Harig JM, Goldstein JL, Layden TJ, and Ramaswamy K. Intestinal distribution of human Na⁺/H⁺ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am J Physiol Gastrointest Liver Physiol 271: G483–G493, 1996.[Abstract/Free Full Text]
13. Gawenis LR, Stien X, Shull GE, Schultheis PJ, Woo AL, Walker NM, and Clarke LL. Intestinal NaCl transport in NHE2 and NHE3 knockout mice. Am J Physiol Gastrointest Liver Physiol 282: G776–G784, 2002.[Abstract/Free Full Text]
14. Geibel JP. Secretion and absorption by colonic crypts. Annu Rev Physiol 67: 471–490, 2005.[CrossRef][ISI][Medline]
15. Geibel JP, Rajendran VM, and Binder HJ. Na⁺-dependent fluid absorption in intact perfused rat colonic crypts. Gastroenterology 120: 144–150, 2001.[CrossRef][ISI][Medline]
16. Hoogerwerf WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, and Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29–G41, 1996.[Abstract/Free Full Text]
17. Ikuma M, Kashgarian M, Binder HJ, and Rajendran VM. Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon. Am J Physiol Gastrointest Liver Physiol 276: G539–G549, 1999.[Abstract/Free Full Text]
18. Krishnan S, Rajendran VM, and Binder HJ. Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon. Am J Physiol Cell Physiol 285: C1246–C1254, 2003.[Abstract/Free Full Text]
19. Ledoussal C, Woo AL, Miller ML, and Shull GE. Loss of the NHE2 Na⁺/H⁺ exchanger has no apparent effect on diarrheal state of NHE3-deficient mice. Am J Physiol Gastrointest Liver Physiol 281: G1385–G1396, 2001.[Abstract/Free Full Text]
20. Masereel B, Pochet L, and Laeckmann D. An overview of inhibitors of Na⁺/H⁺ exchanger. Eur J Med Chem 38: 547–554, 2003.[CrossRef][ISI][Medline]
21. Musch MW, Bookstein C, Xie Y, Sellin JH, and Chang EB. SCFA increase intestinal Na absorption by induction of NHE3 in rat colon and human intestinal C2/bbe cells. Am J Physiol Gastrointest Liver Physiol 280: G687–G693, 2001.[Abstract/Free Full Text]
22. Paimela H, Kiviluoto T, Mustonen H, and Kivilaakso E. Intracellular pH in isolated Necturus duodenal mucosa exposed to luminal acid. Gastroenterology 102: 862–867, 1992.[ISI][Medline]
23. Romero MF. The electrogenic Na⁺/HCO₃^– cotransporter, NBC. JOP 2: 182–191, 2001.[Medline]
24. Schultheis PJ, Clarke LL, Meneton P, Harline M, Boivin GP, Stemmermann G, Duffy JJ, Doetschman T, Miller ML, and Shull GE. Targeted disruption of the murine Na⁺/H⁺ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest 101: 1243–1253, 1998.[ISI][Medline]
25. Schultheis PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na⁺/H⁺ exchanger. Nat Genet 19: 282–285, 1998.[CrossRef][ISI][Medline]
26. Singh SK, Binder HJ, Boron WF, and Geibel JP. Fluid absorption in isolated perfused colonic crypts. J Clin Invest 96: 2373–2379, 1995.[ISI][Medline]
27. Wakabayashi S, Shigekawa M, and Pouyssegur J. Molecular physiology of vertebrate Na⁺/H⁺ exchangers. Physiol Rev 77: 51–74, 1997.[Abstract/Free Full Text]
28. Zachos NC, Tse M, and Donowitz M. Molecular physiology of intestinal Na⁺/H⁺ exchange. Annu Rev Physiol 67: 411–443, 2005.[CrossRef][ISI][Medline]

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