INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IN MAMMALIAN COLON, intracellular pH (pH_i) regulation and electroneutral NaCl absorption are closely related functions. Apical Na⁺/H⁺ exchange is the luminal uptake mechanism contributing to transcellular Na⁺ absorption (3, 38), and its activity consequently affects pH_i in the colonic epithelium (8, 29, 44, 51). Among the six known Na⁺/H⁺ exchanger isoforms, only NHE1, NHE2, and NHE3 have been found in the colonic epithelium on the basis of mRNA expression and immunological detection (4, 5, 18). NHE1 is ubiquitously expressed in mammalian cells to maintain pH homeostasis of cells (57). In epithelial cells of the intestine and colon, NHE1 is localized in the basolateral membrane (4). In contrast, NHE2 and NHE3 have a more restricted tissue distribution and are apical membrane proteins in intestinal epithelia (4, 5, 26, 54, 55). Within the rat colon, immunostaining detected NHE3 exclusively in the surface cells, whereas NHE2 mRNA was detected predominantly on the surface cells with a diminishing gradient that terminated in the upper third of the colonic crypt (4, 5). In human colon, evidence has suggested that NHE2 mRNA is present deeper in the crypts (18).

The physiological role of different Na⁺/H⁺ exchangers is controversial. In NHE3-knockout mice, basal fluid absorption by the intestine was severely diminished (49), demonstrating that NHE3 plays at least a permissive role in overall salt and water absorption. In complementary experiments, NHE2-knockout mice have no obvious intestinal absorption defect (48). Aldosterone increases abundance of NHE3, but not NHE2, in the rat proximal colon (11). However, it is NHE2, and not NHE3, that is responsible for the enhanced Na⁺ absorption in response to a low-Na⁺ diet in chicken colon (17), and it has been reported that rat colonic NHE2 and NHE3 are affected in parallel by Na⁺ depletion (27). In rat proximal colon, evidence suggests that NHE2 is the predominant contributor to basal Na⁺ absorption (7), although the role of NHE3 seems more dominant in isolated membrane vesicles from the same tissue (11, 27). Adding to the complexity, evidence suggests that rat colonic crypts may contain apical Na⁺/H⁺ exchange activity that requires extracellular Cl (44, 45). Cl-dependent function has been proposed as evidence of a new Cl-NHE isoform Na⁺/H⁺ exchanger in the colon, but an intracellular Cl dependence has recently been shown to be a feature of conventional NHE isoforms as well (1).

There is also a controversy about the colonic epithelial cell types contributing to salt and water absorption. In contrast to the classic view of crypts as secretory structures, recent studies have shown that colonic crypts also contribute to fluid absorption (21, 52). Although crypt epithelial cells are less directly exposed to the luminal contents than surface epithelial cells, they are an abundant cell type of the colonic epithelium because of the density and size of crypts in the tissue (23). Therefore, their overall contribution to absorption could be substantial.

The major goal of this study was to question whether apical Na⁺/H⁺ exchange was a feature of the cells near the base of colonic crypts and, if so, to characterize that function with respect to known NHE isoforms and the Cl-dependent NHE that have been reported. We have loaded isolated mouse colonic mucosa with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester (BCECF-AM), a pH-sensitive dye, and measured activity of apical Na⁺/H⁺ exchange by confocal microscopy while retaining normal epithelial architecture in the mucosa. Results demonstrate that even cells in the base of colonic crypts have the potential to contribute to Na⁺ absorption and that known NHE isoforms are sufficient to explain results.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissue preparation. ICR mice (Harlan, Indianapolis, IN) were killed with halothane vapor (Halocarbon Laboratories). The distal colon was excised, flushed with saline, and stripped of muscle layers, as described previously (12). Mucosal sheets were kept in DMEM (GIBCO BRL) on ice and used within 3 h. For experiments, a microscope chamber allowed mounting of mucosal sheets of muscle-stripped mouse colon, such that a physiological saline could be superfused independently at the luminal and serosal surfaces (12). The serosal surface of the tissue was mounted facing the microscope objective lens (Zeiss C-Apo ×40) to facilitate study of crypt epithelium. Dye loading and perfusion were performed on tissue mounted in the microscope chamber. For pH_i measurements, the solution contained 5 µM BCECF-AM (Molecular Probes) in Na⁺ medium [in mM: 130 NaCl, 5 KCl, 1 MgSO₄, 2 CaCl₂, 1 (Na)PO₄, 20 HEPES, 25 mannose, and 1 probenecid, titrated to pH 7.4 with NaOH]. Incubation was at room temperature for 30 min. After BCECF dye loading, the chamber was placed on the microscope stage and continuously perfused.

Perfusate solutions. Perfusate solutions were based on the Na⁺ medium described above. In Na⁺-free solutions, tetramethylammonium (TMA) chloride replaced all NaCl mole for mole; in ammonium media, 25 mM NH₄Cl replaced equimolar NaCl or TMA chloride; and in isobutyrate media, 130 mM sodium isobutyrate or TMA isobutyrate replaced equimolar NaCl or TMA chloride. When perfusates with low Na⁺ concentration were required, NaCl was substituted mole for mole for TMA chloride in TMA medium to give defined Na⁺ concentrations. Two Cl-free media were used: sodium gluconate medium [in mM: 130 sodium gluconate, 5 potassium gluconate, 4 calcium gluconate, 1 MgSO₄, 1 (Na)PO₄, and 20 HEPES] and NaNO₃ medium [in mM: 130 NaNO₃, 5 KNO₃, 2 Ca(NO₃)₂, 1 MgSO₄, 1 (Na)PO₄, and 20 HEPES]. When necessary, Cl-free media were made Na⁺ free by equimolar substitution of the Na⁺ salt by 130 mM TMA gluconate or TMA nitrate, respectively. To inhibit Na⁺/H⁺ exchange activity, 20 µM 5-(N-ethyl-N-isopropyl)amiloride (EIPA; RBI), S-1611, or HOE-694 [3-(methanesulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate; the two latter compounds were generous gifts from Dr. H. J. Lang, Adventis Pharma Deutschland, Frankfurt/Main, Gemany] was added to select media. All perfusate and drug solutions were prepared fresh directly before use, and final perfusates were adjusted to pH 7.4.

Confocal microscopy and image analysis. Images were collected using a Zeiss LSM510 confocal microscope. To measure BCECF fluorescence, excitation was alternated between 488- and 458-nm lines of an argon laser, with emission collected at >505 nm at a single photomultiplier tube detector held at constant gain and dark current during the experiment. The LSM510 confocal microscope allowed rapid switching of excitation wavelength after each scan line for excitation ratio imaging, so that <10 ms separated data collected at the two wavelengths. Ratio images of fluorescence at 488 nm to fluorescence at 458 nm were calculated after subtraction of background images at each wavelength, and values from the entire cytosol of all imaged epithelial cells within a crypt were averaged. To measure pH_i gradients, 1- to 2-µm² regions in subapical and subbasal areas of colonocytes were analyzed as previously described (24, 34). In some experiments, tissue was not loaded with BCECF but was superfused instead with Na⁺ medium containing 100 µM Lucifer yellow [CH lithium salt (LY); Molecular Probes]. The extracellular LY dye was imaged with 458-nm excitation and >505-nm emission. In each image, average LY fluorescence (minus background) was recorded from crypt lumens, lateral intercellular spaces (LIS), and lamina propria tissue adjacent to the crypts. In BCECF and LY experiments, images were routinely collected at the same focal plane over time, with the confocal pinhole adjusted for 1.5-µm optical section thickness. 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. Background images were collected from nontissue areas of the chamber. Post-data-acquisition image analysis was performed (MetaMorph, Universal Imaging) to analyze results from four to six crypts per experiment.

pH_i calibration. To avoid problems with poor permeation of nigericin into tissue (data not shown), intracellular dye calibration was performed with isolated mouse colonocytes (28) using high-K⁺ medium and nigericin (an artificial K⁺/H⁺ exchanger), as described previously (36). Colonocytes were loaded with 5 µM BCECF-AM and superfused with 130 mM K⁺ and 10 µM nigericin solution at pH 6-8 during imaging on the confocal microscope. Excitation ratios of fluorescence at 488 nm to fluorescence at 458 nm at different pH values were obtained (Fig. 1A) from confocal images. A single-site concentration dependency equation was used to fit a pH_i calibration curve by nonlinear regression to data of eight independent preparations (Prism, Graphpad Software; Fig. 1B). The calibration curve demonstrates a favorable dynamic range of the ratio for pH_i measurement and was used to calculate pH_i of experimental data. After each experiment, a solution containing 25 µM BCECF in pH 7 Na⁺ medium was imaged on the confocal microscope stage as an internal standard to normalize results of the nigericin calibration curve to daily settings of the confocal microscope. All averaged results are presented as means ± SE of separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Intracellular pH (pHi) calibration of mouse colonocytes. Fresh isolated mouse distal colonocytes were loaded with 5 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-AM in high-K+ medium with 10 µM nigericin. Cells were superfused with high-K+ medium containing 10 µM nigericin, and pH varied between 6 and 8 during imaging on the confocal microscope stage. Background-corrected excitation ratio of fluorescence at 488 nm to fluorescence at 458 nm was calculated from raw fluorescence images. A: results from a representative experiment showing time course of ratio change in response to changes in medium pH. B: results from 8 experiments (means ± SE) are fit to the pH calibration curve used to determine pHi values in subsequent experiments.

Immunofluorescence staining. Mouse colon was fixed with 2% paraformaldehyde in PBS through cardiac perfusion of the mouse under thiobutabarbital (Inactin, 100 mg/kg) anesthesia. The excised colon was further fixed for 2 h at 4°C in the same fixative. Fixed tissue was rinsed with PBS twice and transferred to 30% sucrose in PBS for 24-36 h at 4°C. The colon was embedded with tissue-freezing medium (EMS). Cryosectioning was done with a microtome cryostat (IEC) at 20°C, and 15- to 20-µm-thick sections were collected on Polysine microscope slides (Erie Scientific). Sections were treated sequentially with PBS (once for 5 min), washing buffer of PBS with 50 mM NH₄Cl (twice for 10 min), blocking buffer of PBS with 50 mM NH₄Cl, 2% BSA, and 0.05% saponin (once for 20 min), primary antibody in blocking buffer (overnight at 4°C), washing buffer (4 times for 5 min), 10 µg/ml secondary antibody (Alexa 488-labeled goat anti-rabbit IgG; Molecular Probes) in blocking buffer (60 min at room temperature), washing buffer (twice for 5 min), nuclear staining with 4 µg/ml propidium iodide in PBS (once for 5 min), and washing buffer (twice for 5 min) and mounted with Prolong Antifade Kit (Molecular Probes). The polyclonal antisera against NHE1 (human NHE1 aa 631-746), NHE2 (rat NHE2 aa 676-813), and NHE3 (rat NHE3 aa 528-648) were produced in rabbits (kindly provided by Drs. E. B. Chang and M. Musch, University of Chicago). NHE1 and NHE3 antisera have been described previously (4, 35). With the use of NHE2-transfected fibroblasts, NHE2 antiserum has been characterized as specific for an 84-kDa protein, competed by the immunizing peptide (M. Musch, personal communication). Antisera to NHE1 and NHE3 were used with 1:100 dilutions and to NHE2 with 1:900 dilution. Preimmune serum from the same rabbits producing antisera was used as control. Slides were imaged on the confocal microscope. Samples were excited with 488 nm, and emission was collected at 500-530 nm (Alexa) and 620-680 nm (propidium iodide). Positive and control slides were imaged with identical confocal settings.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

As described in MATERIALS AND METHODS, muscle-stripped colonic mucosa was mounted in a microscope chamber and examined by confocal microscopy. Our goal was to examine polarized Na⁺/H⁺ exchange function in crypt epithelial cells, but interpreting those experiments required characterization of how well the luminal and serosal perfusates were separated in the perfusion chamber. Specifically, we needed to test whether the epithelium sustained a barrier between luminal and serosal compartments and whether the LIS between adjacent crypt epithelial cells was a site of restricted access to the serosal perfusates. For these general questions, we superfused tissue with the membrane-impermeant fluorescent dye LY in the luminal or serosal compartment and imaged LY fluorescence by confocal microscopy every 10 s while we focused near the base of colonic crypts.

When LY was added to the serosal compartment, LY fluorescence rapidly equilibrated into LIS [half time (t_1/2) = 14 ± 3 (SE) s, n = 6 experiments] and the lamina propria tissue between crypts (t_1/2 = 20 ± 0 s). Some LY bound to collagen fibers in the lamina propria and could not be washed out, but in the majority of lamina propria regions, LY fluorescence was rapidly reversible on removal of LY from the perfusate and washed out with rapid kinetics (t_1/2 = 30 ± 3 s). LY also appeared in crypt lumens (t_1/2 = 16 ± 2 s), demonstrating a transepithelial leak of the dye. As shown in Fig. 2, all fluorescence in LIS and crypt lumens was rapidly and completely reversible on removal of serosal LY (t_1/2 = 11 ± 2 and 13 ± 2 s, respectively). The levels of steady-state fluorescence in the presence of serosal LY are shown in Table 1, with results normalized to the (reversible) fluorescence in lamina propria. Luminal fluorescence was 10% of fluorescence in the lamina propria. Because the LIS are physically smaller than optical resolution (i.e., any imaged pixel includes regions containing LIS and non-LIS regions), fewer dye molecules are available to report fluorescence, and so LIS are dim, even when equilibrated with LY concentrations in the lamina propria [also observed previously with carboxyseminaphthorhodofluor (SNARF)-1 fluorescence] (12). In addition to demonstrating transepithelial leak of LY, results suggest that the rate of fluid entry and exit at the LIS and surrounding lamina propria tissue is rapid and indistinguishable.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Transmucosal permeability of extracellular Lucifer yellow (LY) dye. Tissue in the microscope chamber was superfused with Na+ medium with or without 100 µM LY. Confocal images of tissue were collected over time while tissue was continuously superfused. Superfusates were independently controlled at the luminal (L) or serosal (S) surfaces. Results were compiled separately from the same images for dye accumulation in the crypt lumens and the lateral intercellular spaces (LIS) between adjacent crypt colonocytes. Raw fluorescence values were collected from a representative time course during polarized presentation of LY to the colonic mucosa. Results are representative of 6 preparations.

When added to the luminal compartment, LY slowly equilibrated in the lumen (t_1/2 = 122 ± 12 s, n = 6 experiments; Fig. 2). This is consistent with previous measurements of SNARF-1 fluorescence (13) and shows that fluid in the luminal perfusates only slowly migrates down the crypt lumen to undergo mixing. Even after luminal fluorescence attained steady-state levels, Table 1 shows that negligible fluorescence (i.e., not significantly different from zero in one-sample t-test, P > 0.2) was detected in the LIS and lamina propria (2 and 4% of luminal fluorescence, respectively). Given the known transepithelial leak of LY, defined by the serosal application of dye in the same experiments, results suggest that rapid mixing in the serosal compartment leads to effective control of extracellular fluid composition in this space, despite known transepithelial leakage from the lumen. Conversely, serosally added components will have a more substantial effect to alter composition of the fluid in the crypt lumen because of relatively slow mixing in the luminal compartment. Thus, for studies of polarized functions (e.g., Na⁺/H⁺ exchange) activated by extracellular factors (e.g., Na⁺) in perfusates, results encourage luminal application of those factors to yield the tightest conclusions about the membrane localization of effects.

Visualization of BCECF-loaded colonic mucosa during superfusion. As described in MATERIALS AND METHODS, muscle-stripped colonic mucosa was loaded with BCECF-AM and then continuously superfused with dye-free medium. Figure 3 shows confocal fluorescence images of BCECF-loaded colonic mucosa during superfusion, imaged at 488-nm excitation and >505-nm emission. BCECF loads into crypt epithelial cells as well as nonepithelial cells in the lamina propria surrounding crypts. Because the mucosa was oriented in the chamber with serosal surface adjacent to the objective lens, we could image crypt epithelial cells clearly. The series of images shows the transition between imaging the base of crypts (Fig. 3A) and the crypt opening (Fig. 3C) as focus is advanced in the microscope. In all subsequent experiments, results are reported from the region directly above the base of the crypt, at the point where multiple crypt lumens were visible and individual epithelial cells were aligned along their apical-basal axis in a single focal plane of the image. Because of tissue motion in the dually perfused chamber, the plane of focus could shift along the crypt-surface axis of the crypt during an experiment. The crypt base is used as a landmark to reposition the focal plane as needed during an experiment.

View larger version (71K): [in this window] [in a new window]   Fig. 3.   Confocal images of BCECF-AM-loaded mouse colonic mucosa. Smooth muscle-stripped mouse distal colonic mucosa was mounted into a microscope chamber, loaded with BCECF-AM, and superfused with Na+ medium. Mucosa was imaged with a Zeiss LSM510 confocal microscope at 488-nm excitation and >500-nm emission. Large, circular multicellular structures are colonic crypts imaged in cross section. Series of images were taken at different focal planes at the base of crypts (i.e., nearest the objective lens; A), focused 5 µm deeper into tissue to the midpoint of the basal cells (B), and focused another 5 µm into tissue to reach the crypt lumen (C). Arrowhead points to crypt epithelium, and arrow points to crypt lumen.

Polarized activation of Na+/H⁺ exchangers. With the use of confocal microscopy, images of BCECF-loaded tissue were collected every 1 min, and results were analyzed from crypt colonocytes in the field of view. To activate Na⁺/H⁺ exchange, we used media without added bicarbonate/CO₂ and acidified the tissue by transient exposure to a weak base (25 mM NH₄Cl; ammonia medium) in luminal and serosal superfusates. Simultaneous with ammonium exposure, Na⁺ was removed from the luminal perfusate (substitution with TMA; TMA medium) to allow adequate time to wash Na⁺ from this compartment. On removal of ammonia medium, colonocyte pH_i acidified rapidly (Fig. 4A). During this treatment, Na⁺ was removed from the serosal medium to halt all Na⁺/H⁺ exchange activity. Na⁺ was then selectively returned to the luminal or serosal perfusate to activate apical or basolateral Na⁺/H⁺ exchange, respectively. As shown in Fig. 4A, colonocyte pH_i alkalinized after cells were exposed to luminal Na⁺. To activate NHE activity, we added luminal 140 mM Na⁺, an Na⁺ concentration much higher than the Michaelis-Menten constant of NHEs for Na⁺ (55, 57, 59) . In this case, full activation of apical NHE will occur before the crypt lumen is equilibrated with the perfusate Na⁺ concentration. On the basis of mixing kinetics in the crypt lumen, we predict that even 2 min after addition of luminal Na⁺, the luminal Na⁺ concentration is 70 mM, which should maximally stimulate the transporter. This is reasonably well matched with our rate of data collection (every 1 min) and should allow reliable measures of transport activity to be collected. Subsequent serosal Na⁺ addition elicited a more rapid pH_i recovery to return pH_i to normal resting level. A second round of NH₄Cl exposure, acidification, and pH_i recovery showed that the pH_i recovery was robust and reproducible in our experimental system. Rates of Na⁺-dependent pH_i recovery were calculated for both rounds of acidification by using the lowest common pH_i value from the two recoveries as the starting value for rate calculation: a way to account for the known pH_i sensitivity of Na⁺/H⁺ exchange activity and accommodate a data set in which the level of acidification cannot be perfectly controlled. No significant difference between the first and second acidification was detected in the rates of pH_i recovery in response to luminal Na⁺ (Fig. 4B) calculated from 4 min of pH_i recovery from the lowest common pH_i. Similar calculations were performed for results from the subsequent addition of serosal Na⁺ after subtraction of the rate of apical Na⁺/H⁺ exchange directly before addition of serosal Na⁺ (to estimate rates of basolateral Na⁺/H⁺ exchange). There was no significant difference (P = 0.25) in basolateral Na⁺/H⁺ exchange after the first vs. second acidification (0.25 ± 0.03 and 0.21 ± 0.04 pH/min, respectively, n = 6). These absolute values calculated from (2-3 min) pH_i recovery after serosal Na⁺ should be viewed with reservation, because linear rates of pH_i change were sometimes poorly resolved due to rapid pH_i recoveries.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Luminal and serosal Na+ activate pHi recovery in colonocytes. Values are means ± SE from 4 experiments. A: time course of cellular pHi response in colonic crypt epithelial cells acidified by NH4Cl prepulse and Na+-free superfusion. Luminal Na+ addition activated a pHi recovery. Subsequent serosal Na+ addition accelerated pHi rise toward normal resting level. Data show 2 rounds of acidification and subsequent pHi recovery. TMA, tetramethylammonium. B: pHi recovery rates of initial 4 min after luminal Na+ addition from 1st and 2nd rounds were calculated starting at lowest common pHi between the 2 rounds of acidification.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Polarized Na+ addition: comparison of pHi response between epithelial and nonepithelial cells in the colonic mucosa. Experiments were performed as described in Fig. 4 legend. A-C: pseudocolor images of intracellular BCECF ratio (488 nm/458 nm), with qualitative correspondence to pH change indicated in the color bar. A: cells in mucosa were acidified by NH4Cl prepulse and kept in Na+-free TMA medium. Crypt colonocytes (arrows) and nonepithelial cells in the lamina propria (arrowheads) acidified. B: luminal Na+ addition caused pHi recovery of crypt colonocytes but not lamina propria cells. Note lamina propria cell at top left, directly adjacent to colonic crypt base. C: subsequent serosal Na+ addition caused further pHi rise in colonocytes and initiated pHi recovery in lamina propria cells. D: direct comparison of BCECF fluorescence ratio time course simultaneously recorded from crypt epithelial cells and lamina propria cells in the same experiment. Results are representative of 4 experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   pHi response to different serosal Na+ concentrations: comparison between epithelial and nonepithelial cells in the colonic mucosa. BCECF fluorescence ratio time course simultaneously recorded from crypt epithelial cells and lamina propria cells was directly compared in the same experiment. Tissue was exposed to indicated Na+ concentration in the serosal perfusate while images were collected every 30 s.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Homogeneity of pHi response among crypt colonocytes. pHi from 5 randomly selected colonic epithelial cells in each crypt shown in Fig. 5, A-C, is quantified. Each analyzed colonocyte responded with pHi alkalinization to luminal (Lum) and serosal (Ser) Na+ presentation of 140 mM Na+. Values are means ± SD; n = 20 cells. **P < 0.001.

Apical Na+/H⁺ exchanger is inhibited by EIPA. We first tested EIPA, an inhibitor that blocks a wide spectrum of NHE isoforms, including NHE1, NHE2, and NHE3 (39, 59). The experimental protocol described for Fig. 4 was used, but 20 µM EIPA was added to luminal perfusates to inhibit apical Na⁺/H⁺ exchange activity in the second round of acidification. Results in Fig. 8A qualitatively show that EIPA inhibited pH_i recovery elicited by luminal Na⁺. The pH_i recovery rate was calculated as described for Fig. 4, and results in Fig. 8B show that luminal EIPA inhibited the response to luminal Na⁺ by 79 ± 3.9% (n = 4). We also tested the effect of basolateral EIPA on Na⁺/H⁺ exchange activity of crypt colonocytes. Results (Fig. 9) showed that basolateral 20 µM EIPA inhibited the Na⁺/H⁺ exchange activated by luminal Na⁺, a result requiring EIPA and/or Na⁺ leakage across the epithelium. As noted earlier, serosal addition of substances leads to greater problems defining sidedness of action. Because basolateral Na⁺/H⁺ exchange was also strongly inhibited by EIPA (Fig. 9), we could test for the reversibility after removal of EIPA. As shown in Fig. 9, EIPA inhibition was not readily reversible during 15 min after EIPA removal from perfusates. Given evidence that EIPA is at best slowly reversible, results in Fig. 8 can be evaluated for the inhibition of basolateral NHE as well as the putative apical transporter. In that experimental series, rates of pH_i recovery after addition of serosal Na⁺ (0.31 ± 0.02 pH/min) were significantly less after exposure to EIPA (0.22 ± 0.01 pH/min, n = 4, P < 0.05), although the 28% decrease was only slightly greater than the nonsignificant 20% decrease reported in the absence of drug and clearly distinct from the 79% inhibition in response to luminal Na⁺ activation. Results suggest that the combination of luminal EIPA and luminal Na⁺ is an effective pairing for selective analysis of apical Na⁺/H⁺ exchange and, combined with earlier results, support the presence of distinct apical and basolateral Na⁺/H⁺ exchangers in colonocytes.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Luminal ethylisopropylamiloride (EIPA) inhibits colonocyte Na+/H+ exchange activity in response to luminal Na+. Values are means ± SE from 4 experiments. A: after a 1st round of NH4Cl prepulse, luminal Na+-dependent pHi recovery was activated. After a 2nd round of acidification, 20 µM EIPA was added to the luminal superfusion before luminal Na+ addition. B: pHi recovery rates of initial 4 min after luminal Na+ addition from 1st and 2nd rounds were calculated starting at lowest common pHi between the 2 rounds of acidification. **P < 0.001 vs. control.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Serosal EIPA inhibits colonocyte pHi recovery in response to luminal and serosal Na+ and is poorly reversible. Values are means ± SE from 3 experiments.

Differential effect of isoform-specific NHE inhibitors. To identify candidate NHE isoforms on the apical membrane of crypt epithelium, we compared two NHE inhibitors. To inhibit NHE3, we used S-1611, a compound that has relatively high specificity for inhibition of NHE3 vs. NHE2 (50). Conversely, we used HOE-694, which is relatively specific for inhibition of NHE2 vs. NHE3 (15). Figure 10A shows qualitatively that, in the presence of luminal 20 µM S-1611, the pH_i recovery stimulated by luminal Na⁺ was similar to control, a result confirmed by quantification in Fig. 10B. In contrast, luminal HOE-694 inhibited the pH_i recovery stimulated by luminal Na⁺ by 89 ± 2% (n = 4; Fig. 11). Similar to results with serosal EIPA, serosal 20 µM HOE-694 caused inhibition of apical and basolateral NHE (data not shown), suggesting that the drug was also leaky across the epithelial layer. Results are most consistent with NHE2, but not NHE3, as a candidate for the apical Na⁺/H⁺ exchanger near the base of colonic crypts.

View larger version (15K): [in this window] [in a new window]   Fig. 10.   Luminal S-1611 does not inhibit colonocyte Na+/H+ exchange activity in response to luminal Na+. Protocol described in Fig. 8 legend was used to compare results in the absence with results in the presence of 20 µM luminal S-1611. Values are means ± SE from 4 experiments. A: time course of pHi response in the absence or presence of 20 µM luminal S-1611. B: pHi recovery rates of initial 4 min after luminal Na+ addition from 1st and 2nd rounds were calculated starting at lowest common pHi between the 2 rounds of acidification.

View larger version (15K): [in this window] [in a new window]   Fig. 11.   Luminal HOE-694 inhibits colonocyte Na+/H+ exchange activity in response to luminal Na+. Protocol described in Fig. 8 legend was used to compare results in the absence with results in the presence of 20 µM luminal HOE-694. Values are means ± SE from 4 experiments. A: time course of pHi response in the absence or presence of 20 µM luminal HOE-694. B: pHi recovery rates of initial 4 min after luminal Na+ addition from 1st and 2nd rounds were calculated starting at lowest common pHi between the 2 rounds of acidification. *P = 0.01 vs. control.

Short-chain fatty acids. Short-chain fatty acids (SCFAs) are major anions of the colonic lumen and stimulate apical Na⁺/H⁺ exchange to promote colonic Na⁺ absorption (2, 20, 47). We asked whether luminal SCFA activates the HOE-694-sensitive Na⁺/H⁺ exchanger in crypt colonocytes. The pH_i of crypt epithelial cells was monitored during exposure to luminal 130 mM isobutyrate in the absence and presence of luminal 20 µM HOE-694. Results in Fig. 12 show that isobutyrate acidified crypt epithelial cells and activated an HOE-694-sensitive Na⁺/H⁺ exchanger. HOE-694 inhibited 76 ± 2% (n = 4) of apical Na⁺/H⁺ exchange activated by the SCFA, suggesting that ammonium prepulse and SCFA activate a similar or identical transporter.

View larger version (18K): [in this window] [in a new window]   Fig. 12.   Luminal HOE-694 inhibits isobutyrate-activated colonocyte Na+/ H+ exchange activity. Values are means ± SE from 4 experiments. A: luminal TMA-isobutyrate (T-B) medium acidified crypt epithelial cells, and subsequent switch to sodium isobutyrate (Na-B) medium initiated pHi recovery. Apical Na+/H+ exchange in the absence and presence of 20 µM apical HOE-694 is compared using the protocol described in Fig. 8 legend. B: pHi recovery rates of initial 4 min after luminal Na+ addition from 1st and 2nd rounds were calculated starting at lowest common pHi between the 2 rounds of acidification. *P < 0.01 vs. control.

Testing Cl dependence of crypt apical Na⁺/H⁺ exchange. Recent studies have reported that all apical Na⁺/H⁺ exchange is dependent on extracellular Cl in crypt epithelial cells from rat distal colon (43-45). To test for Cl dependence of apical Na⁺/H⁺ exchange in crypt epithelial cells from mouse distal colon, we measured apical Na⁺/H⁺ exchange with and without Cl (Fig. 13A). The mucosa was first exposed to 25 mM ammonium in Na⁺-free solution. Subsequent removal of ammonium caused rapid cellular acidification. Simultaneously, Cl was removed from the luminal and serosal superfusion (gluconate substitution) for 8-10 min. Returning luminal Na⁺ activated pH_i recovery in the absence of Cl, and subsequent luminal Cl addition did not accelerate the pH_i recovery rate. To test for effects of different anion substitution, Cl was replaced by gluconate (Fig. 13A) or nitrate. Control experiments with the continuous presence of Cl were used for comparison with Cl-free conditions. Results from four experiments using each condition are shown in Fig. 13B, which shows that Na⁺/H⁺ exchange activity is similar in the presence of Cl, gluconate, or nitrate. Results indicate that any apical Na⁺/H⁺ exchange in mouse distal colonic crypt epithelium is independent of extracellular Cl.

View larger version (14K): [in this window] [in a new window]   Fig. 13.   Crypt colonocyte Na+/H+ exchange is not dependent on extracellular Cl. A: representative time course experiment showing crypt colonocytes acidified by ammonium prepulse and then exposed to Na+-free and Cl-free TMA-gluconate (TMA-G) medium. After pHi acidified to a steady state, luminal Na+ was returned to luminal Cl-free superfusion [sodium gluconate (Na-G) medium] to activate pHi recovery. Further addition of Cl to the luminal superfusion did not accelerate pHi recovery. B: compiled results comparing pHi recovery rates of initial 4 min mediated by return of luminal Na+ in Cl-free conditions (NaCl replaced by sodium gluconate or NaNO3) with rates in the presence of Cl throughout the experiment (i.e., using conventional TMA medium and Na+ medium). Rates were calculated from the same starting pHi. Results under the 3 conditions were not significantly different (P > 0.05, n = 4 experiments in each condition).

Testing for pH_i gradient in crypt colonocytes. Using polarized HT29-C1 cells, we had observed more extreme pH_i changes in the subapical cytoplasm than in the region adjacent to the basal pole of the cell. This led to pH_i gradients being observed in HT29-C1 cells in response to luminal SCFA exposure or NH₄Cl prepulse (24, 34). Because the apical-basal axis of the epithelial cells was directly imaged in our native tissue experiments, we asked whether a similar pH_i gradient could be observed in mouse crypt colonocytes under comparable conditions. We analyzed small (1-2 µm²) regions of cytoplasm and compared the pH_i reported at the subapical and subbasal domains of crypt epithelial cells. In Fig. 14, these subcellular pH_i values in the resting state (exposed to Na⁺ medium), in the presence of luminal SCFA (absence of Na⁺ in all superfusates), and after NH₄Cl prepulse (also in the absence of Na⁺ in all superfusates) are compared. As shown in Fig. 14, pH_i was not significantly different between the two subcellular sites under any of the tested conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 14.   Testing for pHi gradients in crypt colonocytes. Measurements from subcellular regions were made in cells exposed to Na+ medium (resting pH, n = 8) or in Na+-free conditions during exposure to luminal isobutyrate (+isobutyrate, n = 3) or Na+-free conditions after transient exposure to NH4Cl medium (post NH4, n = 5).

Immunostaining of NHE isoforms. Figure 15 shows immunofluorescent staining of mouse distal colon with polyclonal antisera raised against the divergent COOH-terminal sequences of NHE1, NHE2, and NHE3. With the use of nuclear staining to mark cellular structures, results show that NHE1 was expressed on the basolateral membrane of epithelial cells along the crypt-surface axis (Fig. 15, a and b). In contrast, NHE3 was expressed on surface but not crypt epithelial cells (Fig. 15, g and h), and the majority of NHE3 was in the apical or subapical domain of the surface cells. The expression of NHE2 in the colon was more complex: it was observed at the apical region of the surface epithelial cells (Fig. 15d) and also in crypt epithelial cells (Fig. 15, d and e). NHE2 stained the apical membrane of cells near the base of the crypts but, similar to NHE3, also displayed cytosolic staining. For all antisera, preimmune serum confirmed specificity of the signal. Results suggest that NHE2, or another NHE isoform with similar epitopes, is a candidate for the apical exchanger near the base of colonic crypts.

View larger version (119K): [in this window] [in a new window]   Fig. 15.   Immunofluorescent localization of NHEs in mouse distal colon. Red, nuclear staining with propidium iodide; green, NHE immunofluorescence (see MATERIALS AND METHODS for staining protocols). *, Crypt lumen; arrow, surface epithelium. Images are for NHE1 (a-c), NHE2 (d-f), and NHE3 (g-i). Images a, d, and g exposed crypt-to-surface view of the colonic epithelium. Images b, e, and h are cross sections of crypts near the crypt base. Images c, f, and i are controls using the same dilution of preimmune sera as the primary antisera. Images are representative of results from 3 animals. Scale bars, 20 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study introduces the use of BCECF, the most commonly used fluorescent pH_i indicator, for ratiometric measurements of living native tissue by confocal microscopy. Recent advances in commercial instrumentation allow rapid switching between appropriate excitation wavelengths (458 and 488 nm) for the use of excitation ratio imaging while minimizing concern about motion artifacts in living specimens. Using the method in conjunction with a chamber that allows separate superfusion of the luminal and serosal compartments of isolated colonic tissue (12), we have applied the method to analyze functional properties of the Na⁺/H⁺ exchangers expressed in the crypt epithelium.

Experiments analyzed the fidelity of the preparation for selective presentation of substances to the luminal vs. serosal compartments and the apical vs. basolateral membrane of the colonocytes. In the serosal compartment, Na⁺ and the structurally unrelated LY dye had rapid access to nonepithelial cells in the lamina propria and basolateral membrane of colonocytes. No evidence was found for slow or restricted mixing of the serosal perfusate in the LIS between colonocytes compared with the directly adjacent lamina propria tissue. Others and we previously observed that pH in the LIS of cultured epithelia and colonocytes can be distinct from that in the serosal tissue and is relatively constant in response to changes in acid/base conditions (9, 12, 25, 34). This was shown to be due to fixed proton buffers on LIS membranes, rather than altered ion diffusion in the LIS (19, 25, 33, 58). This model is supported by our observation of rapid fluid exchange between the lamina propria and the LIS. Our results suggest that LIS Na⁺ concentration can be rapidly altered, even by low concentrations of added Na⁺, suggesting lack of substantial Na⁺ binding or buffering in LIS. Of importance for studies in which Na⁺ is added to luminal or serosal perfusates, results demonstrated that the basolateral membrane of colonocytes was exposed to a fluid that was rapidly and efficiently refreshed by serosal perfusates.

In contrast, multiple results suggested that the crypt lumen has only slow access to luminal perfusates and is thereby subject to greater influence from leakage of fluid and substances from the serosal compartment. The most direct demonstration is that luminal equilibration of an extracellular marker (LY) added to the luminal perfusate required sixfold more time than serosal equilibration of the same marker added to the serosal perfusate. We also observed a marked asymmetry when the transepithelial LY leakage between the bulk fluid environments of the crypt lumen and the lamina propria was compared as the orientation of LY transepithelial gradients was reversed. In response to serosal LY, 10% of the dye concentration appeared in the crypt lumen. The response to luminal LY addition was significantly less (P = 0.026) dye accumulation in the lamina propria, at only 4% (and this value was not significantly different from zero). This suggests that the extracellular fluid in the serosal compartment of the colonic mucosa is more rapidly and efficiently controlled by the perfusate than the extracellular fluid in the luminal compartment.

Experiments unequivocally demonstrated limited leakage of an extracellular fluid marker (LY, 450 g/mol) between the serosal and luminal compartments. Additionally, experiments showed that NHE inhibitors (EIPA and HOE-694) and/or Na⁺ could also traverse the mucosa. Our results place limits on how leakage of Na⁺ across the mucosa could confound the conclusion that apical Na⁺/H⁺ exchange mediates the response to luminal Na⁺. On the basis of our resolution of pH_i recovery in lamina propria cells (Fig. 6), the amount of luminal 140 mM Na⁺ reaching the serosal tissue adjacent to colonocytes must be <3 mM. To explain the rapid rate of colonocyte Na⁺/H⁺ exchange activated by luminal Na⁺ as due to erroneous presentation of Na⁺ at basolateral membranes, the LIS/basal membranes would have to be exposed to a much higher Na⁺ concentration: 10 mM Na⁺ at basolateral membranes. This more than threefold Na⁺ gradient over 10-30 µm is clearly incompatible with results being due to 1) gross mixing of the two perfusates via a poorly built microscope chamber or 2) transepithelial Na⁺ absorption at the surface epithelium adding Na⁺ to the serosal tissue around crypts. The only tenable explanations are those in which Na⁺ appears at the basolateral surface by flux directly across the crypt epithelium. One possibility is a substantial transcellular Na⁺ flux that loads the LIS/basal extracellular region with Na⁺. This Na⁺ absorptive function requires a robust apical Na⁺ uptake route in the crypt base cells to continually fuel Na⁺ loading in these well-mixed extracellular spaces, and the apical route would have to be EIPA and HOE-694 insensitive and a transport reaction other than Na⁺/H⁺ exchange. The only other apical transport mechanism contributing to mammalian Na⁺ absorption is Na⁺ channels, and evidence suggests that these are solely expressed at the surface epithelium (31). The second possibility is a paracellular Na⁺ flux locally activating LIS Na⁺/H⁺ exchangers. Because of the localized entry of Na⁺ across tight junctions and rapid fluid mixing in the LIS, a gradient of decreasing Na⁺/H⁺ exchange activity along the apical-basal axis of the LIS is predicted. No gradient of pH or Na⁺ concentration has been observed along the apical-basal axis of the LIS (9, 10, 25, 32). In contrast, our observations would require a steep Na⁺ gradient characterized by <3 mM Na⁺ near the basal pole of colonocytes and an average 10 mM Na⁺ along the length of the LIS. Although such a gradient may seem unlikely, LY leakage ensures some paracellular Na⁺ flux. Results suggest that the crypt colonocyte tight junctions must be relatively impermeable to Na⁺, since transepithelial accumulation of the 450 g/mol LY is greater (4% on the basis of results in Table 1) than the maximal accumulation of Na⁺ that is predicted from study of lamina propria cells (<2% on the basis of results in Fig. 6). In our analysis of the apical Na⁺/H⁺ exchange, we have optimized conditions for selectively detecting apical transport by adding Na⁺ and inhibitors only to the luminal compartment. In addition to the studies described above that evaluate the influence and accessibility of perfusates to the spaces surrounding colonocytes, the suitability of this approach is suggested by experiments with EIPA. Luminal application of EIPA produces only slight inhibition of basolateral NHE (presumptive NHE1) compared with apical NHE, despite using an EIPA concentration (20 µM) that is ~1,000-fold greater than the inhibition constant (K_i) of NHE1 for the inhibitor (39, 59) and 100-fold greater than the IC₅₀ in the presence of 145 mM Na⁺ (42). For all the reasons cited above, we believe that our analysis has resolved an Na⁺/H⁺ exchanger localized to the apical membrane of crypt colonocytes.

Absorptive transporter in relatively undifferentiated colonic crypt epithelium. In mouse colonic crypts from distal colon, epithelial cells near the base of colonic crypts are proliferating and are only separated by a few cell divisions from the stem cells positioned at the crypt base (6, 41). Although our experiments are clearly not studying the stem cells, the expression of more differentiated functions (such as absorptive transporters) is believed to start nearer the colonic surface. With the use of isolated, microperfused crypts from rat distal colon, it has been observed that crypts (in the absence of secretory stimuli) absorb fluid in an Na⁺-dependent manner and express an (Cl-dependent) apical Na⁺/H⁺ exchange (21, 43-45, 52). In these experiments, the need to cannulate the upper portion of the crypt and puncture/cannulate the crypt base allowed analysis in the midregion of the crypt. In contrast, our measurements preserve normal epithelial architecture while allowing study of colonocytes directly adjacent to the crypt base. In this site, we also observe apical Na⁺/H⁺ exchange and report that this transport can be activated by physiological luminal stimuli such as Na⁺ and SCFAs. These results strongly suggest that even cells near the base of the crypt express an absorptive-type transporter and, for this reason, have at least the potential to contribute to Na⁺ absorptive function. Given the high abundance of the Cl secretory channel cystic fibrosis transmembrane conductance regulator in the lower crypt (56) and the observation that all cells near the crypt base express apical Na⁺/H⁺ exchange (Fig. 7), we speculate that individual crypt epithelial cells in the colon may mediate Na⁺ absorption and Cl secretion.

Candidate NHEs at the crypt base. Recent studies on isolated, microperfused rat distal colonic crypts and apical membrane vesicles have shown a Cl-dependent Na⁺/H⁺ exchange activity (43-45). Using protocols designed to closely parallel those applied previously to resolve Cl-NHE (44, 45), we were unable to demonstrate any Cl dependence of apical Na⁺/H⁺ exchange in mouse distal colonic crypts. We compared a relatively permeant (nitrate) and impermeant (gluconate) anion substitution with no difference in results. The discrepancy between our results and previous studies may be due to species differences (rat vs. mouse) or site of analysis (crypt base vs. midcrypt region). It should be noted that known NHE isoforms can be Cl dependent in some cases due to an (incompletely understood) effect of intracellular Cl (1). However, our results clearly demonstrate that the Cl-NHE as previously analyzed in rat distal colonocytes is not a ubiquitous function of colonic crypt epithelia.