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Am J Physiol Cell Physiol 278: C973-C981, 2000;
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Vol. 278, Issue 5, C973-C981, May 2000

pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers

Djikolngar Maouyo1, Shaoyou Chu2, and Marshall H. Montrose2

1 Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21205; and 2 Department of Physiology and Biophysics, Indiana University, Indianapolis, Indiana 46202


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

In the colonic mucosa, short-chain fatty acids change intracellular pH (pHi) and extracellular pH (pHe). In this report, confocal microscopy and dual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were used for direct evaluation of pHi and pHe in a simple model epithelium, HT29-C1 cells. Live cell imaging along the apical-to-basal axis of filter-grown cells allowed simultaneous measurement of pH in the aqueous environment near the apical membrane, the lateral membrane, and the basal membrane. Subapical cytoplasm reported the largest changes in pHi after isosmotic addition of 130 mM propionate or 30 mM NH4Cl. In resting cells and cells with an imposed acid load, lateral membranes had pHi values intermediate between the relatively acidic subapical region (pH 6.3-6.9) and the relatively alkaline basal pole of the cells (pH 7.4-7.1). Transcellular pHi gradients were diminished or eliminated during an induced alkaline load. Propionate differentially altered pHe near the apical membrane, in lateral intracellular spaces between adjacent cells, and near the basal membrane. Luminal or serosal propionate caused alkalinization of the cis compartment (where propionate was added) but acidification of the trans compartment only in response to luminal propionate. Addition of NH4Cl produced qualitatively opposite pHe excursions. The microscopic values of pHi and pHe can explain a portion of the selective activation of polarized Na/H exchangers observed in HT29-C1 cells in the presence of transepithelial propionate gradients.

carboxyseminaphthorhodofluor-1; laser-scanning confocal microscopy; epithelium; polarity; NHE1; NHE2; colon; short-chain fatty acid; propionate; ammonium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

OVER THE LAST 15 years, evidence has accumulated that epithelial cells sustain microenvironments of pH both outside and inside their plasma membranes. Many laboratories have shown that extracellular pH (pHe) near the plasma membrane can be distinct from the pH of the bulk medium presented to the gastric epithelium (2, 11, 30, 35), cultured renal epithelia (5, 17), or colonic epithelium (8, 19, 23, 31). In many of these cases, pHe was perturbed by physiological stimuli, and microenvironment pHe was generated at least in part by epithelial transport activity. More limited information is available about intracellular pH (pHi) microenvironments in the cytosol, but such reports indicate that pHi heterogeneity can result from membrane transport of acid/base equivalents and/or could have a potential role in regulating cellular acid/base transport (20, 21, 39).

In the colon, pH microenvironments have been proposed as an important factor allowing the tissue to mediate efficient sodium absorption. In the colonic lumen, propionate, acetate, and butyrate [collectively termed short-chain fatty acids (SCFAs)] are the major anions (4). These SCFAs, generated from bacterial fermentation of unabsorbed carbohydrates and proteins, act to stimulate electroneutral sodium absorption by activating apical Na/H exchange in colonocytes (3, 18, 34). Although Na/H exchange activation is often assumed to be due to pHi acidification by the weak acid SCFAs, it has recently been shown that bulk cytosolic pHi cannot explain sodium transport activation in isolated colonic tissue (12) or the HT29-C1 cultured colonocyte model (33).

This has led investigators to question anew whether local changes in pH are an essential part of SCFA action in the colon. Initially, investigators used macro- and micro-pH electrodes to study the pHe microclimate at the colonic epithelial surface, but the response to SCFAs was controversial (23, 31). The advent of new optical approaches and fluorescent indicators has provided tools to visualize dynamic events near or at the membrane surface of living cells. Near-membrane pH sensors and confocal microscopy of extracellular dyes have recently resolved changes in pHe near the apical surface of surface and crypt colonocytes in response to SCFAs (7, 8, 19). In colonic crypt epithelia, evidence suggests that the lateral intercellular spaces (LIS) between adjacent cells have an acidic pHe that was not measurably perturbed by luminal SCFA (7). In contrast, the lamina propria tissue surrounding crypts demonstrated changes in pHe on luminal or serosal SCFA addition (7, 8, 10). This raised the possibility that epithelial cells may be surrounded by three distinct pHe microenvironments at the apical surface, LIS, and basal surface.

To unravel mechanisms for regulation of intracellular and extracellular microenvironments and understand the impact of such mechanisms on cellular function, it has become important to measure the extent of changes in pHi and pHe in the same experimental system. For that reason, this report evaluates the local changes in pHi and pHe in polarized monolayers of HT29-C1 cells. This human colonic cell line has previously reported a selective activation of apical and basolateral Na/H exchangers (NHEs) (NHE2 and NHE1, respectively) that could not be explained by changes in bulk pHi (20, 33). Furthermore, measurements of pHi heterogeneity alone were not sufficient to explain this selective activation of polarized NHEs (20). By incorporating measurements of pHe and study of the lateral membrane region, we now find that part of the selective NHE activation can be predicted. Furthermore, by qualitatively comparing the response to an SCFA with that of the weak base ammonium, we can start to consider the specificity of mechanisms that lead to these local pH changes.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Tissue culture. HT29-C1 cells were grown as described previously (20, 33). Briefly, cells were grown under 5% CO2-95% air atmosphere in DMEM containing 25 mM glucose. For most experiments, cells were trypsinized and seeded onto Anotec filters (Whatman) attached to a support made of two fused plastic coverslips (Fisher Scientific) in which 0.063- to 0.094-in. holes had been punched. Cell monolayers grown over the hole had direct access to medium at the basolateral membranes, and only confluent monolayers over the holes were studied. Cells were examined 2-3 days postconfluency over the hole. For experiments that did not require polarized presentation of bathing medium, cells were seeded onto glass coverslips. HT29-C1 cells were used for experiments from passages 9-18.

Microscope perfusion chamber. A previously described microscope chamber (28) was utilized for mounting cells grown on filters or glass coverslips. A modification was needed to permit imaging of cells on filters with the C-Apo ×40 water-immersion objective (with only 220 µm of working distance). As described above, two coverslips were fused with organic solvents as a glue (1:1 cyclohexanone:chloroform). In this manner, cell monolayers seeded on the upper coverslip would be on an elevated surface, ~250 µm closer to the objective than in the absence of the extra coverslip thickness. In addition, acrylic tape (Furon, New Haven, CT) was attached to the chamber surface to slightly (~50 µm) deepen the perfusion space. The result was a 200-µm net repositioning of cells, which reproducibly placed the monolayers within the working distance of the ×40 objective yet allowed an unobstructed superfusion over the apical membrane. For clarity, superfusion over the apical membrane is hereafter referred to as "luminal" superfusion and superfusion over the basolateral membrane is referred to as "serosal" superfusion.

Superfusion solutions. Cells in the microscope chamber were continuously superfused during experiments. All media were based on "NaCl medium" [containing (in mM) 130 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, and 20 HEPES and titrated to pH 7.4 with NaOH]. In NH4Cl and sodium propionate media, 30 or 130 mM NaCl was replaced mole for mole by NH4Cl or sodium propionate, respectively. To calibrate the intracellular carboxyseminaphthorhodofluor (SNARF)-1 response, cells were exposed to a high-potassium medium [containing (in mM) 20 HEPES, 20 MES, 75 KCl, 35 potassium gluconate, 14 sodium gluconate, 1 CaCl2, 1 MgSO4, and 2 tetramethylammonium chloride and titrated from pH 6 to 8 with tetramethylammonium hydroxide]. In these experiments, cells were loaded with SNARF-1-AM in high-potassium medium that contained 10 µM nigericin at pH 8.0, and all subsequent perfusion solutions contained 3 µM nigericin as pH was varied in the high-potassium medium (28).

Confocal pH measurement. To measure pHi, cells were loaded for 30 min with 10 µM SNARF-1-AM before study. In separate experiments to measure pHe, all superfusates contained 0.1 mM SNARF-1 free acid. The method for collection of confocal images and image analysis has been described previously (7, 20). Briefly, SNARF-1 fluorescence emissions at 550-600 and 620-680 nm were simultaneously collected in two channels of a confocal microscope (model LSM410, Zeiss) in response to 488-nm argon ion laser (Omnichrome) excitation. Approximately 3% of maximum laser power was used for imaging, which did not produce measurable photobleaching of intracellular SNARF-1 dye after scanning to collect 100 images (data not shown). Images were collected with a C-Apo ×40 water-immersion objective, with eight line averages to reduce noise. For all measurements of pH, images were collected along the apical-to-basal axis of the cells (i.e., x-z images collected perpendicular to the focal plane of the microscope). After subtraction of background values (from regions without dye), fluorescence emission ratios (ratio of 620-680 nm to 550-600 nm) were calculated and calibrated to pH values, as described in RESULTS (Metamorph software, Universal Imaging). Low values of fluorescence were eliminated (masked by thresholding) in raw fluorescence images to eliminate edge effects when ratio images were subsequently calculated. A pH 7.0 extracellular dye calibration was performed to standardize instrument response daily.

Several regions of interest (ROIs) were measured within images to explore the changes in pH near different membrane domains. In x-z images collected along the apical-to-basal axis of cells, raw fluorescence images provided more information about cell structure than ratio images. Therefore, ROIs were positioned using raw fluorescence images for orientation and measurements subsequently made from the identical location in the corresponding ratio image. ROIs were positioned to measure pHi or pHe (depending on the site of dye loading) from the ~3-µm space adjacent to the apical boundary of cells. Similarly, ROIs were positioned to report pH from the ~3-µm space adjacent to the basal boundary of cells at the filter growth support. ROIs were also positioned to measure pH near the lateral membrane of cells. To measure pHe near the LIS between adjacent cells, the majority of the LIS was quantified in any given image. The pHi at the lateral membrane was measured in a 1 × 5 µm ROI placed adjacent to the lateral membrane, at the midpoint of cells (only those cells with the entire apical-to-basal axis present in the images were used). The long axis of this ROI was positioned parallel to the direction of the lateral membrane.

Statistics. Values are means ± SE. Statistical evaluation was by Student's t-test (single comparisons) or by repeated-measures ANOVA, with significance of individual comparisons determined by Bonferroni multiple comparisons test. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Our first goal was to appraise whether SNARF-1 could be used to comparatively measure pHi and pHe. A laser-scanning confocal microscope (model LSM410, Zeiss) was used to provide 488-nm illumination that excited SNARF-1, a pH-sensitive fluorescent dye, in epithelial monolayers of HT29-C1 cells grown on filters. As shown previously (20), cell cytosol could be loaded with SNARF-1 via exposure to the membrane-permeant precursor SNARF-1-AM (Fig. 1, A and B), and the outline of individual cells was readily identified by dye exclusion from the LIS between adjacent cells viewed along the apical-to-basal axis (Fig. 1A) or in a cross section across the middle of the monolayer (Fig. 1B).


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  		Fig. 1.   Confocal fluorescence of intracellular and extracellular carboxyseminaphthorhodofluor (SNARF)-1 in HT29-C1 monolayers grown on permeable membrane filters. Cells were superfused continuously with NaCl medium while images of confocal fluorescence at 550-600 nm were collected. A and B: cells loaded intracellularly with SNARF-1-AM. A: image collected perpendicular to plane of membrane filter, with cells imaged along apical-to-basal axis. B: image collected in plane parallel to membrane filter, with focus at midpoint of monolayer. C and D: cells exposed to membrane-impermeant SNARF-1 free acid. C: image collected perpendicular to plane of membrane filter. Arrows, lateral intercellular space between adjacent cells that span entire cell layer. D: image collected in plane parallel to membrane filter, with focus at midpoint of monolayer. In all image orientations and dye conditions, it is possible to define boundaries of individual cells. L, luminal; S, serosal.

Monolayers could also be examined during superfusion with SNARF-1 free acid. As shown in Fig. 1, C and D, the membrane-impermeant SNARF-1 free acid was excluded from cells. Extracellular spaces were sites of dye loading, including the LIS between individual cells and the space at the junction between the basal pole of the cell and the filter. In intracellular and extracellular SNARF-1 images, LIS were commonly observed to be continuous across the cell layer, confirming the presence of a single cell layer (arrows in Fig. 1C). In intracellular and extracellular dye imaging, the cell monolayer was observed to vary from ~20 to 40 µm in height among preparations. Results reported here were similar, independent of cell height.

To integrate pH values reported by extracellular or intracellular SNARF-1, we compared the sensitivity of fluorescence emission ratios of extracellular and intracellular SNARF-1 to changes in pH. We observed (Fig. 2) that intracellular SNARF-1 had a potentially smaller dynamic range than extracellular SNARF-1, as measured in the confocal microscope. Using high potassium and nigericin in the medium to control pHi of cells grown on glass coverslips (28), the intracellular SNARF-1 response was used to calibrate pHi measurements in subsequent experiments. SNARF-1 in solution was used to calibrate pHe measurements. Separate experiments were used to measure pHi or pHe because of the difficulty of balancing both signals in a single experiment and the loss of structural information that occurred when dye was present in all imaged spaces.


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  		Fig. 2.   Intracellular and extracellular SNARF-1 calibration curves. Intracellular dye response () was measured using cells that had been pH clamped with high-potassium nigericin (see MATERIALS AND METHODS), with medium pH set to values indicated. Extracellular dye response (open circle ) was measured on confocal microscope stage in drops of NaCl medium at indicated pH. Values were normalized to response at pH 7.0 to facilitate comparison between curves. Values are means ± SD of 13-20 measurements.

pHi. Confocal microscopy was used to evaluate spatial heterogeneity of pHi along the apical-to-basal axis of HT29-C1 cells, via direct imaging along this axis, as shown in Fig. 1A. Experiments compared the response of cells to propionate or ammonium in isosmotic superfusates.

Propionate acidified all regions of HT29-C1 cytosol independent of the polarity of addition, but evaluation of subcellular pHi values suggested that pHi varied regionally within cells. Figure 3 shows results from a time course experiment, analyzing results from the entire monolayer (whole cell response; Fig 3A), or from three defined subcellular regions adjacent to different plasma membrane domains (Fig. 3B). The three subcellular regions were adjacent to 1) the apical membrane facing the luminal perfusate, 2) the basal membrane near the filter growth support, or 3) the lateral plasma membrane between adjacent cells (analyzed at the midpoint between the apical and basal limits of the cell monolayer but at the lateral edge of the cell; see MATERIALS AND METHODS for details about size and location of these subapical, subbasal, and lateral domains). Similar to results in Fig. 3, we observed differences in the magnitude of pHi overshoots in response to polarized SCFA addition in colonic epithelium, which are explained in that tissue by activation of (high-activity basolateral) NHE1 by serosal but not luminal SCFA (27). We previously showed that because acid extruders (e.g., NHEs) change pH, they drive intracellular accumulation of SCFAs, and therefore removal of medium propionate causes alkaline overshoots (9).


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  		Fig. 3.   Time course of intracellular pH (pHi) response to propionate medium in a representative experiment. Cells were loaded intracellularly with SNARF-1 and superfused continuously at luminal and serosal compartments. Superfusates were composed of NaCl medium (Cl) or propionate medium (Prop). Confocal images were collected along apical-to-basal axis of cell monolayer. A: values derived from average ratio measured from entire imaged monolayer at each time point. This approximates whole cell response that would have been determined under nonconfocal conditions. B: separate analysis of subapical domain, within 3 µm adjacent to apical boundary of cell (black-down-triangle ); subbasal domain, within 3 µm adjacent to basal boundary of cell (black-triangle); and lateral domain, adjacent to lateral cell membrane at midpoint of cell (open circle ). Each point is mean from 10-20 cells in monolayer.

Although we analyzed subapical and subbasal domains previously (20), we did not attempt to correlate results near the lateral membrane. As shown in Fig. 3B and in compiled results of such experiments in Table 1, pHi values at the lateral membrane were intermediate between those at the subapical and subbasal domain, suggesting that events leading to the observed pHi heterogeneity were not identical along the entire basolateral membrane. Furthermore, Table 1 shows changes in the magnitude of the intracellular proton gradient (analyzed as change in pHi) between the apical and basal poles under different experimental conditions. The change in pHi across the cells in the presence of luminal or serosal propionate was significantly greater (P < 0.05) than that after removal of serosal propionate. The measurements reported in Table 1 were time averaged (3-5 min) to reduce noise in small subcellular regions, but approximate the peak pHi excursions observed after a solution change because of the slow recovery of pHi in the presence of propionate (Fig. 3) (20). For this reason, results can include influence from pHi recovery mechanisms, although such pH changes are small in this time frame vs. the initial pH excursions we seek to approximate.

                              
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  		Table 1.   Effect of SCFA on pHi in HT29-C1 monolayers

Visual inspection of the results in Table 1 suggests that, in response to any polarized addition or removal of propionate, the change in pH reported in the subapical portion of the cell was greater than the change in pHi measured at the subbasal portion of the cell. When the subapical and subbasal changes in pHi observed in individual monolayers are paired, the apical change in pHi was statistically larger (P < 0.05) for each solution change in Table 1, except during addition of serosal propionate (P = 0.06).

NH4Cl was used to make a complementary evaluation about effects of a weak base vs. the weak acid propionate. We found that 30 mM NH4Cl also perturbed subcellular pHi homeostasis. As shown in the representative time course experiment in Fig. 4 and the compiled results summarized in Table 2, luminal or serosal addition of 30 mM NH4Cl perturbed average pHi of the monolayer (Fig. 4A) by virtue of effects that manifested differently in the apical, lateral, and basal portions of the cytosol (Fig. 4B). Similar to propionate, addition or removal of NH4Cl caused changes in pHi that were most pronounced in the subapical domain, and lateral pHi was intermediate between subapical and subbasal values. Because addition of propionate or NH4Cl will drive pHi in opposite directions, addition of the weak base was predicted to decrease the transcellular change in pHi, in contrast to the increase observed with propionate. This trend was observed (Table 2); however, 30 mM NH4Cl had less striking effects than 130 mM propionate during addition or removal of the perturbing compound.


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  		Fig. 4.   Time course of pHi response to NH4Cl medium in a representative experiment. Cells were loaded intracellularly with SNARF-1 and superfused continuously at luminal and serosal compartments. Superfusates were composed of NaCl medium (Cl) or NH4Cl medium (NH4Cl). Confocal images were collected along apical-to-basal axis of cell monolayer. A: average ratio measured from entire imaged monolayer at each time point, to approximate whole cell response. B: separate analysis (see Fig. 3B legend) of subapical domain (black-down-triangle ), subbasal domain (black-triangle), and lateral domain (open circle ). Each point is mean from 10-20 cells in monolayer.


                              
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  		Table 2.   Effect of NH4Cl on pHi in HT29-C1 monolayers

Results in Table 2 were analyzed for differential effects of NH4Cl on subapical vs. subbasal pHi. Addition or removal of luminal NH4Cl caused a significantly greater subapical than subbasal change in pHi (P < 0.05), but there was no corresponding statistical difference in the change in pHi detected in the two regions in response to serosal NH4Cl (P > 0.09).

pHe. Previous work suggested that localized change in pHe around colonocytes is also a component of weak acid and base action (7, 8, 10, 19). Therefore, HT29-C1 cells were superfused with media containing the membrane-impermeant SNARF-1 free acid to measure pHe.

Figure 5 presents results from an individual time course experiment reporting pHe adjacent to the apical membrane and within the LIS when a cell monolayer is exposed to propionate. The pHe in both spaces is affected by propionate. Results from a series of such experiments are shown in Table 3, and measurements of basal pHe are included for comparison. The most potent stimulus to induce pHe changes was luminal propionate addition, which significantly alkalinized pHe at the apical surface (P < 0.01 in paired comparisons) while simultaneously acidifying pHe in the LIS (P < 0.001) and at the basal surface (P < 0.05). Removal of luminal propionate caused a reversal of effects at the surface and LIS (P < 0.001). For all other transitions initiated by superfusate changes, the LIS pHe changed (P < 0.01), but no other space manifested a statistically significant change. Briefly, polarized addition of propionate caused pHe alkalinization in the cis compartment (i.e., the same side as propionate addition) and, in some cases, simultaneously caused acidification of the trans compartment. Results were qualitatively similar to observations across the epithelium of mouse colonic crypts (7, 8, 10).


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  		Fig. 5.   Time course of extracellular pH (pHe) response to propionate medium in a representative experiment. Cells were continuously superfused with media containing 0.1 mM SNARF-1 at luminal and serosal compartments. Superfusate was NaCl or propionate medium. Confocal images were collected along apical-to-basal axis of cell monolayer, and pHe was calculated in spaces adjacent to epithelial cells. Two different extracellular regions were separately analyzed in same images: near apical domain of luminal superfusate within 3 µm adjacent to apical boundary of cells () and lateral intercellular spaces between adjacent HT29-C1 cells (open circle ). Each point is mean from assigned regions within entire imaged monolayer.


                              
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  		Table 3.   Effect of SCFA on pHe in HT29-C1 monolayers

Given the modest changes in pHe recorded in these experiments, one trivial explanation could be that pH of experimental solutions was not well controlled before the experiment. However, all superfusates were carefully titrated to pH 7.40 ± 0.02 before experiments. In addition, if the same solutions were run through the microscope chamber in the absence of cells (with a cell-free plastic coverslip), then pHe was accurately recorded as 7.4 (data not shown). This suggests that the observed pHe changes are the result of cellular acid/base transport.

Similar experiments were performed using ammonium to determine whether the responses reported above were specific to a weak acid and/or an SCFA. Table 4 presents measurements from exposure of cells to luminal or serosal 30 mM NH4Cl. Statistical comparisons revealed that although luminal NH4Cl was able to reversibly acidify the apical surface (P < 0.01), no other effects on pHe were significant.

                              
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  		Table 4.   Effect of NH4Cl on pHe in HT29-C1 monolayers


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

Propionate is one of the physiological SCFAs that can alter pHi and pHe as part of their action in the colon (7, 8, 13, 14, 19, 23, 38). The most prominent role ascribed to SCFA-induced pH change is stimulation of electroneutral sodium absorption in the colon via activation of apical Na/H exchange in colonocytes (3, 18, 34). We previously used the HT29-C1 cell as a model colonocyte to study the mechanism for NHE activation by propionate. This is based on results showing that HT29-C1 cells and colonocytes share at least one apical NHE isoform, NHE2 (16, 20, 24, 26), as well as the basolateral NHE1 (16, 20). Furthermore, in the presence of a luminal SCFA, bulk cytosolic pHi cannot explain sodium transport activation in isolated colonic tissue or in the HT29-C1 cultured colonocyte model (12, 33).

In summary of earlier reports from experiments with HT29-C1 cells, luminal propionate preferentially stimulates activity of the apical NHE2, such that NHE2 activity is 3.8-fold greater than the activity of the basolateral NHE1 (20, 33). Conversely, serosal propionate preferentially activates basolateral NHE1 fourfold compared with apical NHE2. We concluded earlier that local changes in pHi were present but were insufficient to explain selective activation of these polarized NHE isoforms (20).

Activation of polarized NHE isoforms. In this report, a major goal was to ask whether the combined effects of an SCFA on local changes in pHe and pHi could explain this selective activation of the polarized NHE isoforms. On the basis of equations described in the APPENDIX, we have estimated the relative activation of apical NHE2, lateral NHE1, and basal NHE1 via changes in pH. Results are summarized graphically in Fig. 6 and described below.


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  		Fig. 6.   Model predictions for selective activation of Na/H exchange isoforms NHE1 and NHE2 by propionate-induced pH heterogeneity in HT29-C1 monolayer. A: resting cells in presence of NaCl medium. B: cells exposed to luminal propionate medium. C: cells exposed to serosal propionate medium. Schematics of epithelial cells are drawn with apical membrane to left. Across top half of each cell diagram, pHi and pHe (derived from Tables 1 and 3) are shown at 3 different membrane domains. On the basis of calculations described in the APPENDIX, activity of NHE2 was estimated and is presented in black ovals as a fraction of maximal transport velocity for that transporter. Similar calculations were performed for NHE1, with separate calculations for activity at lateral and basal membrane domains. NHE1 results are presented in stippled ovals as a fraction of maximal transport velocity of NHE1 at the indicated membrane domain.

On the basis of the model calculations of transport activation by pH, an unstimulated cell at rest uses 4.3% of maximal NHE2 activity in the apical membrane, and NHE1 activity will be <1% of maximal in the lateral and basal membranes (Fig. 6A). Previously, we showed that stimulation of HT29-C1 cells by luminal propionate would activate apical NHE 3.8-fold more than basolateral NHE (33). The kinetic model (cf. Fig. 6, A and B) predicts that luminal propionate would activate NHE2 5.5-fold (0.235/0.043) and lateral membrane NHE1 4.5-fold (0.036/0.008), yielding only a 1.21-fold selective activation of NHE2 over NHE1 predicted at lateral membranes (5.5/4.5). A similar calculation predicts that luminal propionate would cause a 1.49-fold activation of NHE2 over NHE1 at the basal membrane. Conversely, serosal propionate stimulated a fourfold activation of basolateral vs. apical NHE in the HT29-C1 cells (33). In the model (cf. Fig. 6, A and C), the predicted activation of NHE1 over NHE2 was 2.45-fold at lateral membranes and 0.8-fold at basal membranes. These calculations suggest that local changes in pH can predict selective activation of NHE isoforms at opposing membrane surfaces; however, only a fraction (<60%) of the selective activation of polarized NHE activity is explained by the model, and NHE1 in the lateral membrane may undergo a transition in activity different from NHE1 at the basal membrane.

Because only about one-half of the observed NHE activation can potentially be explained by the model and our microscopic values of pHi and pHe, there is clearly a need to account for the remaining transport activation. This may require improving the sophistication of the model (which makes a number of simplifying assumptions; see APPENDIX), measuring pH nearer to the membrane (19), and/or recognizing different factors regulating NHE activity in the presence of propionate. In the latter case, it is important to consider that changes in cell volume (15, 32, 37), chloride (38), and possibly intracellular calcium levels (1, 6) may occur as part of exposure to propionate and will affect NHE or other acid/base transporters. Although these factors require further investigation, they are not a concern for the analyses we have performed here. We have used exactly the same ionic conditions to measure surprising changes in NHE activation (20, 33), and our goal here was to question whether localized pH changes under these same conditions could explain these previous observations. Now that we know that aqueous pH changes are not able to explain all the polarized NHE activation, other potential mechanisms have ascended in importance for further study.

Significance of LIS pH. As described above, the LIS may play a crucial role in NHE1 activation. Previous observations in Madin-Darby canine kidney cells and in colonic crypt epithelium are consistent with our observation in HT29-C1 cells that the LIS is an acidic environment (5, 7, 22). However, unlike prior observations in the colonic crypt, propionate significantly affected pHe in the LIS (7). This may imply a fundamental difference in regulation of the LIS pHe between cell types. Differences in pHe regulation in LIS of LLC-PK1 and Madin-Darby canine kidney cell lines have been noted (22). Alternatively, differences may be explained by the inability to mimic complex epithelial architecture in culture. Independent of explanation, it is clear that in cultured and native tissue any basolateral membrane transport or membrane enzyme that is sensitive to pH (e.g., NHE1 and nonionic diffusion of SCFAs or ammonium) will display anomalous behavior with respect to the pH of the bathing medium.

When propionate or ammonium is added to the monolayer, it is difficult to know whether measured pHe results are due to paracellular and/or transcellular flux of the weak acid and base under study. On the basis of observations in guinea pig, SCFA fluxes across tight junctions are nonexistent or occur only in the uncharged form (40). The latter case would also be consistent with our observations of LIS pHe, which could occur due to nonionic diffusion across the tight junction. At this stage, our working hypothesis is that the dominant effects are due to transcellular acid/base transport on the basis of two observations. First, significant changes in pHi are observed in response to the weak acid/base, which can only occur as a result of transmembrane flux across the cell membrane. Second, the tight junction must be responsible physiologically for maintaining a large standing pHe gradient between the luminal and LIS domains, which would be difficult to sustain if large acid/base fluxes occurred across the tight junction.

Transcellular gradients of pHi. We previously observed that a subapical region in HT29-C1 cells is the predominant site at which pHi changes are observed in response to propionate (20). In this report, we find that a structurally unrelated weak base (ammonium) can also preferentially affect pHi in the subapical domain under some conditions. This suggests that the hypothetical subapical H-tight domain proposed by Dagher et al. (12) may also be responsive to ammonia/ammonium. Ammonium had smaller effects than propionate on pHi and pHe, likely because of the lower concentration of NH4Cl (30 mM) than propionate (130 mM) in the experiments. Unfortunately, it was not possible to raise NH4Cl concentrations further without changing medium osmolarity (if sodium was kept constant) or changing cellular sodium content (which occurs in HT29 cells when medium sodium concentration was <90 mM; flame photometry data not shown). At this point, we have no firm idea of the physical basis for the functional compartmentalization of the cytoplasm reported by SNARF-1. Our working hypothesis is that results will be partially explained by an asymmetric distribution of fixed buffers in the cytosol, potentially because of asymmetric distribution of structures (e.g., microfilaments, vesicles) displaying these charges/buffers on their cytosolic surfaces.

The marked stability of basal pole pHi under different conditions can be misleading. Solely on the basis of this observation, it would have been reasonable to conclude that transport (e.g., of propionate) across the basolateral membrane was not driving/mediating large local fluxes of acid/base equivalents. However, there is compelling evidence for a robust flux of acid/base equivalents across the basolateral membrane based on the large changes in pHe in the LIS directly outside the same membrane and the simultaneous large changes of pHi in the subapical intracellular domain after exposure to serosal propionate or ammonium. The acidic pHe of the LIS also presents a large inward gradient of protons that must be maintained across the basolateral membrane. It may not be a coincidence that the basal pole of the cell has basic pHi and a very acidic extracellular environment. Measurements of pHe, therefore, allow us to conclude that the basal pHi of HT29-C1 cells must be robustly defended rather than passively set.

Overall, these results show that pH heterogeneity exists in the aqueous environment near apical and basolateral membranes. Under many circumstances, the apical membrane had an acidic pHi at the cytoplasmic face in combination with an alkaline pH at the extracellular face. Conversely, the basolateral membrane had an alkaline pHi at the cytoplasmic face and an acidic extracellular environment. This pH heterogeneity results in transepithelial and transcellular pH gradients, which may provide a driving force for paracellular and/or transcellular transport. As an example, we have estimated that local and polarized changes in pHi and pHe can partially explain the selective activation of NHEs that is needed to mediate efficient colonic sodium absorption. In summary, the complexity of cellular pH regulatory machinery requires an understanding of microscopic changes in pH and encourages a reappraisal of our understanding of acid/base transport in the epithelial environment.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

On the basis of published values for kinetic parameters for NHE1 [extracellular KT(Na) = 10 mM, Ki(H) = 10-7 M] and NHE2 [extracellular KT(Na) = 50 mM, Ki(H) = 1.26 × 10-7 M] from Yu et al. (41), it was possible to calculate the extent of activation of both exchangers when pHe varied at the apical, LIS, and basal poles of the cells. This was done using a simple model in which extracellular protons are a competitive inhibitor of Na+ activation kinetics (36), as has been shown to occur for NHEs (29). In this case
<FR><NU><IT>V</IT></NU><DE><IT>V</IT><SUB>max</SUB></DE></FR> = <FR><NU>[Na<SUP>+</SUP>]/<IT>K</IT><SUB>T</SUB>(Na)</NU><DE><FENCE>1 + <FR><NU>[Na<SUP>+</SUP>]</NU><DE><IT>K</IT><SUB>T</SUB>(Na)</DE></FR> + <FR><NU>[H<SUP>+</SUP>]</NU><DE><IT>K</IT><SUB>i</SUB>(H)</DE></FR></FENCE></DE></FR> (1)
where [Na+] and [H+] are Na+ and H+ concentrations, respectively, V is velocity of transport, and Vmax is maximal transport velocity. Results were calculated as the fraction of Vmax (V/Vmax), with values of pHe from Table 3, with the assumption that NHE2 was apical, NHE1 was basolateral [as suggested from previous work (20)], and [Na+] was 130 mM. The results of this calculation estimate the extracellular component of NHE transport activation by pHe alone.

Similarly, other kinetic parameters for NHE1 (intracellular K'[H+] = 0.37 µM, Hill coefficient = 2.3) and NHE2 (intracellular K'[H+] = 0.14 µM, Hill coefficient = 1.9) from Levine et al. (25) made it possible to use the Hill equation to calculate the extent of activation (V/Vmax) of NHE isoforms at the values of pHi reported at the apical, lateral, and basal poles of the cell in Table 1. In this case
<FR><NU><IT>V</IT></NU><DE><IT>V</IT><SUB>max</SUB></DE></FR> = <FR><NU>[H<SUP>+</SUP>]<SUP><IT>n</IT></SUP></NU><DE><IT>K</IT>′[H<SUP>+</SUP>] + [H<SUP>+</SUP>]<SUP><IT>n</IT></SUP></DE></FR> (2)
where n is the Hill coefficient. The result of this calculation estimates the intracellular component of NHE transport activation by pHi only.

If it is assumed that 1) extracellular Na+ binding and intracellular H+ binding are independent events and 2) membrane translocation of the ions was rate limiting to NHE activity, transport activity should be approximated by the fractional amount of NHE carrier that is bound to the substrates. The NHE carrier will complete a transport cycle (Na+ transported in and H+ transported out) at a rate that is limited by the aggregate availability of loaded carriers. For this reason, the aggregate action of the intracellular and extracellular components was estimated by multiplying the values derived from Eqs. 1 and 2 by use of physiological pH values depicted in Fig. 6 (and taken from Tables 3 and 1, respectively). Calculated values are presented in Fig. 6 (within the ovals). This estimate of fractional transport activity is an oversimplification but serves as a rough estimate of NHE activation at each membrane domain.


    ACKNOWLEDGEMENTS

This work was performed in the Center for Epithelial Disorders at Johns Hopkins University. This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1 DK-42457 awarded to M. H. Montrose, with a supplement grant awarded to D. Maouyo.


    FOOTNOTES

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.

Address for reprint requests and other correspondence: M. H. Montrose, Medical Sciences 307, 635 Barnhill Dr., Indiana University School of Medicine, Indianapolis, IN 46202-5120 (E-mail: mmontros{at}iupui.edu).

Received 6 July 1999; accepted in final form 23 November 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
APPENDIX

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Am J Physiol Cell Physiol 278(5):C973-C981
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