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Am J Physiol Gastrointest Liver Physiol 276: G259-G270, 1999;
0193-1857/99 $5.00
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Vol. 276, Issue 1, G259-G270, January 1999

Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid

Tamas Gonda, Djikolngar Maouyo, Sharon E. Rees, and Marshall H. Montrose

Departments of Medicine and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205


    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 µM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 µM, basolateral ID50 = 0.3 µM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.

SNARF-1; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; laser scanning confocal microscopy; epithelium; polarity; propionate; NHE1; NHE2; NHE3


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SHORT-CHAIN FATTY ACIDS (SCFAs) are produced in the lumen of the large intestine by bacterial fermentation of the nonabsorbed carbohydrate and protein that progresses into the colonic lumen. There is a large transepithelial gradient of SCFAs across the colonic epithelium under physiological conditions because these monocarboxylates (e.g., acetate, propionate, and butyrate) accumulate to a steady-state level of >100 mM in the colonic lumen but are at <0.5 mM in blood (13). Absorbed SCFAs stimulate salt and water absorption, and their metabolism will generate 7-10% of body energy reserves (3). The mechanism of colonic SCFA absorption is controversial but is coincident with intracellular and extracellular pH changes in the colonic mucosa (5, 7, 8, 10, 14, 16, 47, 48). Present evidence suggests that nonionic and carrier-mediated SCFA transports will directly contribute to the changes in pH (5, 9, 24, 31, 38, 42).

SCFAs stimulate electroneutral sodium absorption up to fivefold in human colon (44), and the model of SCFA-stimulated sodium absorption is tightly linked to the ability of SCFA fluxes to change pH. SCFAs stimulate colonic sodium absorption via activation of apical Na/H exchange. It is commonly accepted that SCFAs cause this activation by changing pH, in simple models by the cellular acidification that occurs when these weak acids are taken up by nonionic diffusion. It is known that multiple isoforms of the NHE family of Na/H exchangers are expressed in the colonic mucosa. These include the NHE2 and NHE3 isoforms that are expressed predominantly in epithelial cells as well as the virtually ubiquitous NHE1 isoform that is found in the basolateral membrane of epithelial cells (50, 51). In addition, both apical and basolateral Na/H exchange activities have been measured in colonocytes (17, 19, 20, 37, 39, 40). At this point, the specific apical NHE isoform or isoforms that mediate electroneutral sodium absorption in the colon have not been confirmed. Identification of these isoforms is important because each has a characteristic response to activation by protons and sodium (51), which will dictate tissue response to the physiological stimulus of SCFA-induced pH change.

Because colonocytes can express apical and/or basolateral Na/H exchangers, it has been unclear how SCFA-stimulated changes in pH could mediate selective activation of apical vs. basolateral Na/H exchange to facilitate efficient sodium absorption. Results from native tissue suggest that this does happen because luminal but not serosal SCFAs can stimulate sodium absorption (1, 21, 36). Previous experiments demonstrated that SCFAs could preferentially activate either apical or basolateral Na/H exchange in HT29-C1 cells (a cloned epithelial cell line derived from a colon carcinoma) (46). This action required transepithelial gradients of SCFAs and was not due to an allosteric effect of SCFAs on the Na/H exchangers. In an apparent paradox, results suggested that changes in pH were the mechanism underlying SCFA effects, but measurements of intracellular pH (pHi) could not resolve the reason for differing effects of transepithelial SCFA gradients (46).

On the basis of these results in HT29-C1 cells, we hypothesized that localized changes in pH near the plasma membrane were affecting local activation of Na/H exchangers. Subsequent experiments in native tissue demonstrated that changes in extracellular pH occurred near the crypt epithelium and were part of SCFA effects that should contribute to polarized activation of Na/H exchange (7). Such observations were consistent with some, but not all, prior observations of a pH microclimate at the colonic surface that was affected by SCFAs (25, 41). Recently, it has been proposed that pHi microenvironments may also be an important regulator of sodium absorption in colonic epithelium (4, 14, 15). This possibility is consistent with the prior observation of pHi gradients in a variety of cell types, using either optical or electrophysiological methods (22, 23, 34, 43). It is also consistent with all prior measurements of pHi, which were averaged from the entire cytosol and colonocyte could not resolve pHi gradients (14, 15, 46).

The goals of the current work are to define the NHE isoforms that mediate polarized Na/H exchange in the HT29-C1 model and to use confocal microscopy to question whether transcellular pHi gradients explain how SCFA gradients selectively activate apical vs. basolateral Na/H exchange. Confocal microscopy has the spatial resolution to resolve subcellular events and is applicable to study of living cells (35). Results show that multiple NHE isoforms are present and that subcellular pHi heterogeneity can only partially explain how SCFAs selectively activate polarized exchangers.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Tissue culture. HT29-C1 cells obtained from D. Louvard (Paris, France) were used between 10-19 passages after original subcloning from the parental HT29-18 line. HT29-C1 cells are stably differentiated when grown in DMEM containing 25 mM glucose, as described earlier (32, 45, 46). In some experiments, cells grown on plastic flasks for 1 wk were suspended using 0.005% trypsin and then exposed to 7 mg ovomucoid trypsin inhibitor (Sigma) and dispersed into a single cell suspension for study by fluorometry (45). For substrate-attached cell experiments, HT29-C1 cells were grown for 3-7 days (1-2 days postconfluency) on permeant membrane filters (Anotec, Whatman) that were attached to plastic frames (Ref. 46 or as slightly modified by D. Maouyo, S. Chu, and M. H. Montrose, unpublished observations, for confocal microscopy).

Fluorometric measurement of pHi. Either suspended cells or confluent cell monolayers on filters were studied. Cells were incubated for 45 min at 37°C with 2 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM; Molecular Probes) in "NaCl medium" [containing, in mM, 130 NaCl, 5 KCl, 20 HEPES, 25 mannose, 1 (Na)PO4, 2 CaCl2, and 1 MgSO4, titrated to pH 7.4]. After dye was loaded, suspended cells were centrifuged 10 s at 2,000 g, resuspended in the absence of dye, and then placed in a stirred fluorometer cuvette thermostated at 37°C (45). After filter-grown cells were dye loaded, the filter was mounted in a fluorometry chamber that allowed independent control of apical and basolateral superfusion (30). BCECF fluorescence was measured in an SLM 500C spectrofluorometer at 520- to 540-nm emission in response to alternating excitation of 500 and 440 nm. The fluorescence excitation ratio (500 to 440 nm) was calibrated vs. pHi as previously described (30, 45, 46).

Confocal microscopy measurement of pHi. Confluent cells on filters were incubated with 10 µM carboxy SNARF-1-AM (Molecular Probes) for 45 min at room temperature. The filters were then mounted in a microscopy chamber placed on the stage of a Zeiss LSM410 confocal microscope and continuously superfused (6) during imaging with a ×40 C-Apo water-immersion objective. Intracellular SNARF-1 was excited with a 488-nm argon laser light, and two fluorescent emissions were collected simultaneously at 550-600 nm and 620-680 nm. During experiments, cells in the monolayer were scanned along the apical-to-basal (xz-plane) axis using eight line averages (4 s per image), and an image was collected once per minute. Results were analyzed postacquisition using Metamorph software (Universal Imaging), Microsoft Excel, and GraphPad Prism. Background-corrected emission ratios (620-680 nm to 550-600 nm) were used to estimate pHi, based on daily calibration of SNARF-1-free acid on the microscope stage (6). Thresholding of raw fluorescence images was used to exclude regions of low fluorescence during analysis. Image analysis was used to estimate pHi either from the entire cell or from five subcellular regions distributed equally along the apical-to-basal axis. Subcellular regions were 4-µm-diameter circles (79 pixels each) that were equally spaced 1-2 µm apart to span the entire apical-to-basal axis of single cells. An example of region placement is shown in Fig. 6A.

Superfusate solutions. All superfusates were based on standard NaCl medium. In SCFA medium, 130 mM chloride was replaced with equimolar propionate. All sodium was replaced with equimolar tetramethylammonium (TMA) in sodium-free TMA-chloride medium. All media were isosomotic (Wescor 5500 osmometer), and all solutions were titrated to pH 7.4. HOE-694 (a generous gift of Dr. H. Lang at Hoescht Marion Roussel) was solubilized in NaCl medium. For the experiments with SNARF-1, all perfusion solutions contained 1 mM probenecid to inhibit dye loss.

PCR. As described previously, total RNA was isolated from confluent flasks of HT29-C1 cells using guanidinium thiocyanate and centrifuging the cell lysate through a CsCl cushion (32). First-strand cDNA was transcribed with Moloney murine leukemia virus-RT (GIBCO BRL) using random hexanucleotide primers (Pharmacia). Unless noted, cDNA templates were subjected to 30 cycles of amplification with recombinant Taq DNA polymerase (Perkin Elmer Cetus) and an annealing temperature of 56°C, and then an aliquot (3 µl) of reaction product was used as template to initiate a second 30-cycle PCR with the same primers. NHE1 primers were 5'-AAGTGTCTGATAGCTGGC-3' and 5'-TGCTCCGCATCATGATGC-3'. NHE2 primers were 5'-AAACATGCCATAGAGATGGC-3' and 5'-CCACCTCATTCTTCCATTC-3'. NHE3 required two rounds of PCR with nested primers. The first round used 5'-GAGAGAAAATGTCAGCGC-3' and 5'-GCAGGAAGGAGTCCACG-3'. Three microliters of this PCR product were used as template for the second round of NHE3 amplification. The nested primers for the second 30 cycles of amplification were 5'-AGGACATGGTCACGCACC-3' and 5'-GGAGAGTAGGGAATCTGC-3', with a 58°C annealing temperature. Caco-2 RNA was a neighborly gift from C. H. C. Yun.

Statistics. Averaged results are presented as means ± SE. Comparison between two groups was performed by unpaired two-tailed t-test, and comparisons among multiple groups were performed by one-way ANOVA with the Bonferroni multiple comparison test (Prism software, GraphPad). Differences of P < 0.05 were considered significant. When multiple comparisons are discussed, only the most conservative level of significance is cited to simplify presentation.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Evidence for multiple NHE isoforms in HT29-C1 cells. We had previously observed both apical and basolateral Na/H exchange in HT29-C1 cells (46), but the molecular identity of these transporters has not been established. To find out whether multiple members of the NHE gene family of Na/H exchangers play a role in pHi regulation of HT29-C1 cells, we examined effects of the NHE inhibitor HOE-694 on suspended cells. HOE-694 has widely different potency for inhibition of NHE1, NHE2, or NHE3 (12). Suspended cells provided a system in which all plasma membrane proteins were equally available to the extracellular environment, albeit in the absence of cellular polarity. As performed previously (45, 46), cells were loaded with a pH-sensitive fluorescent dye (BCECF), and Na/H exchange was measured as the sodium-dependent recovery of pHi from an acid load induced by transient exposure to NH4Cl.

We examined the effects of varying concentrations of HOE-694 added during the pHi recovery from an acid load. Drug inhibition was calculated as the percent change in the linear rate of pHi recovery after vs. before HOE-694 addition. Results of individual runs are shown in Fig. 1; nonlinear least squares curves fit to the data assumed either one or two binding sites for HOE-694. Statistical comparison of the two fits (Prism software, GraphPad) indicated that the two-site model was a better fit to the data (P < 0.005), which suggested that at least two kinetically distinct transporters were responsible for pHi regulation. On the basis of prior work showing that NHE3 had an HOE-694 ID50 = 650 µM (12), the complete inhibition of Na/H exchange by 10 µM HOE-694 suggested that NHE3 was unlikely to contribute to pHi regulation.


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  		Fig. 1.   HOE-694 concentration dependency for inhibition of Na/H exchange. Intracellular pH (pHi) was measured in suspended HT29-C1 cells using 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as described in METHODS. Na/H exchange was quantified as the sodium-dependent recovery from an acid load induced by 30 mM NH4Cl prepulse (45). Linear rates of pHi recovery were compared before vs. immediately after addition of indicated concentrations of HOE-694. Extent of inhibition was calculated as the percent decrease in the rate of pHi recovery by the drug, and each point represents a single determination with results compiled from 3 experiments. Results were fit by 2 equations: a 1-binding site model (dotted line) and a 2-binding site competition model (solid line). Data deviated from the 1-site model (P < 0.05) but not from the 2-site model (P = 0.37). The 2-site model was a significantly better fit (P = 0.005), even accounting for the larger number of fit variables compared with the 1-site model (Prism software, GraphPad). Inset key presents the best-fit parameters derived from both models.

Experiments were performed to determine whether kinetically distinct Na/H exchangers segregated to the apical vs. basolateral membranes. Confluent HT29-C1 cell monolayers on filters were studied in a fluorometry chamber, which allowed independent control of luminal and serosal superfusion (bathing the apical and basolateral membranes, respectively). Cells were acidified by NH4 prepulse, and then sodium was added unilaterally to initiate pHi recovery. During pHi recovery, varying concentrations of HOE-694 were superfused to the same cell surface to measure drug sensitivity. Figure 2 shows results from a single experiment, qualitatively demonstrating differences between sensitivity of Na/H exchange to 1 µM HOE-694 added at either the apical or basolateral membrane. Figure 3 compiles results from five experiments to quantify the inhibitory potency of HOE-694. Curve fitting of results at the apical or basolateral membrane gave the best fit with only a single HOE-694 inhibitor site vs. a two-site model, suggesting that only one Na/H exchange was kinetically resolved at either membrane domain. The dotted line in Fig. 3 is the best fit of the basolateral data set to a two-site model having the HOE-694 affinities defined from suspended cells. This special case of the two-site model is clearly a poor fit. Results suggest the presence of two Na/H exchangers in HT29-C1 cells: one at the apical membrane having a low-affinity HOE-694 binding site and one at the basolateral membrane with a higher-affinity HOE-694 binding. The low-affinity value for suspended cells is indistinguishable from the apical membrane value and is similar to the ID50 reported for NHE2 (12). The high-affinity site value for suspended cells differs by sixfold from the basolateral membrane value, but both most closely resemble the ID50 reported for NHE1 (12).


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  		Fig. 2.   Differing potency of HOE-694 against apical and basolateral Na/H exchange. pHi was measured in filter-grown HT29-C1 cells using BCECF and a conventional fluorometer as described in METHODS. Results from a single monolayer during 2 separate recoveries from an acid load induced by 30 mM NH4Cl prepulse are shown. After cells were equilibrated in tetramethylammonium (TMA)-chloride medium (TMA), NaCl medium (Na) was added to either the luminal or serosal superfusate to selectively activate apical or basolateral Na/H exchange, respectively. At the indicated times, 1 µM HOE-694 was added to the same side of the monolayer as the sodium. As shown, apical Na/H exchange was more resistant to inhibition by HOE-694 than basolateral Na/H exchange.


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  		Fig. 3.   HOE-694 concentration dependency for inhibition of polarized Na/H exchangers. Results were compiled from 5 experiments such as that shown in Fig. 2, using different concentrations of HOE-694 to separately quantify inhibition of apical (black-triangle) or basolateral (bullet ) Na/H exchange. Inhibition of Na/H exchange was measured as described in Fig. 1. Each data point is mean of 3-5 determinations. Results were fit by a 1-site model (solid line) but would not converge with a 2-site model having full freedom to determine 50% inhibitory dose (ID50) values (because only 1 site was needed to fit the curve; the other site was indeterminate). If ID50 values were fixed at 0.05 and 1.6 µM (values determined from suspended cells) and the 2-site model was applied to the basolateral data set, best-fit parameters ascribed 52% of transport to the high-affinity site, although the curve fit (dotted line) was inferior. Key shows the best-fit parameters derived from the 1-site model. These and all averaged results are presented as means ± SE.

To complement these functional analyses, PCR was used to detect the presence of mRNA for specific NHE isoforms. Primers were designed to amplify the most divergent portion of the isoforms, the COOH-terminal region (51). As shown in Fig. 4, RT-PCR detected the presence of NHE1 and NHE2 but not NHE3. The identity of amplified fragments was confirmed by digestion with rare-cutting (6-bp recognition) restriction enzymes predicted from published human NHE sequences. The NHE2 restriction sites were commonly predicted from the human NHE2 sequence of Ramaswamy et al. (18) (GenBank no. S83549) and Ghishan et al. (11) (GenBank no. 81591). The significance of negative NHE3 results in HT29-C1 was confirmed by positive amplification of NHE3 from Caco-2 cells, another human colon carcinoma line that expresses NHE3 several weeks postconfluency (29). Both kinetic and molecular analyses support the lack of NHE3 and the presence of basolateral NHE1 and apical NHE2 in HT29-C1 cells.


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  		Fig. 4.   RT-PCR analysis of NHE isoforms present in HT29-C1 cells. Isoform-specific primers were used to amplify COOH-terminal sequences from NHE1, NHE2, and NHE3 mRNA. PCR products were run on agarose gels and visualized with ethidium bromide. Restriction enzymes confirmed the identity of the amplified fragments. A: cDNA from HT29-C1 was used to amplify fragments of NHE1 or NHE2, as indicated. Predicted 621-bp NHE1 amplification product was observed (lane 1) and had the predicted restriction sites for Ban I (lane 2) and Apa I (lane 3). Similarly, 517-bp NHE2 amplification product (lane 4) had the predicted restriction sites for Hind III (5' 340-bp fragment and 3' 177-bp fragment) (lane 5) and Tth III (5' 162-bp fragment and 3' 355-bp fragment) (lane 6). B: NHE3 primers were used to amplify cDNA from Caco-2 or HT29-C1 cDNA as indicated. Despite use of nested PCR and attempts with several primer pairs, we could not observe a clean PCR product. However, cDNA from Caco-2 cells demonstrated the most prominent band at the predicted 549 bp size (lane 1, marked by arrow) that was cut by Sal I to generate the predicted fragments (160 and 389 bp) (lane 2, marked by *) and by Nco I to generate the correct fragments (264 and 285 bp) (lane 3, marked by >). In contrast, the PCR fragments amplified from HT29-C1 (lane 4) did not give the predicted Nco I fragments (lane 5).

Regulation of pHi gradients by SCFAs and Na/H exchange. Heterogeneity of pHi might explain why luminal-to-serosal transepithelial gradients of SCFAs are required to observe selective stimulation of apical Na/H exchange (4, 14, 15, 46). Therefore, confocal microscopy was used to resolve pHi at a subcellular level. Because luminal SCFA preferentially activates apical Na/H exchange activity, and serosal SCFA preferentially activates basolateral Na/H exchange (46), we compared these two conditions as those predicted to yield the most widely different pHi gradients.

We first tested the sensitivity of confocal microscopy to report pHi recovery of the entire cell cytosol, when imaging along the apical-to-basal pole using the SNARF-1 fluorophore. As shown in the abbreviated time course of Fig. 5A, cells acidified after exposure to serosal 130 mM sodium propionate at time zero and mounted a pHi recovery over the ensuing 14 min in the continuous presence of the SCFAs. Figure 5B shows that the pHi recovery is inhibited by serosal addition of 10 µM HOE-694 or bilateral removal of sodium from the medium. Figure 5C shows pHi recovery in response to luminal 130 mM sodium propionate added at time zero, which examined the same time course as in Fig. 5A. Figure 5D shows that the response to luminal propionate is inhibited by luminal addition of 10 µM HOE-694 or bilateral removal of sodium. These results validate the sensitivity of confocal microscopy to detect activation and inhibition of apical or basolateral Na/H exchange in response to SCFA gradients. Results after HOE-694 addition also suggest apical Na/H exchange is responsible for the majority of total Na/H exchange following stimulation by luminal SCFA, and, conversely, basolateral Na/H exchange predominates after serosal SCFA. Both results are consistent with prior observations that the orientation of the SCFA gradient determines the preferential activation of polarized Na/H exchangers (46).


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  		Fig. 5.   Response to short-chain fatty acids (SCFA) reported in whole cell confocal analyses. Confocal microscopy was used to image cell monolayers loaded with carboxy SNARF-1, as described in METHODS. Cells were directly imaged along the apical-to-basal axis (xz-images) during superfusion with different solutions, and pHi was estimated from the fluorescence of the entire visualized cytosol. Results are presented for 4 time points: 2 min before SCFA addition while cells were in NaCl medium (-2 min), at the nadir pHi directly following SCFA addition (defined as time 0), and 7 and 14 min after time 0 in the continued presence of SCFA. A: cells were exposed to serosal 130 mM sodium propionate while NaCl medium was maintained in the luminal superfusate [n (no. of experiments) = 8]. As shown, confocal microscopy detects pHi recovery in the presence of SCFA. B: cells were exposed to serosal 130 mM TMA-propionate in combination with luminal TMA-chloride (down-triangle, n = 3) or serosal sodium propionate plus 10 µM HOE-694 in combination with luminal NaCl medium (triangle , n = 5). C: cells were exposed to luminal 130 mM sodium propionate while NaCl medium was maintained in the serosal superfusate (n = 6). D: cells were exposed to luminal 130 mM TMA-propionate in combination with serosal TMA-chloride (down-triangle, n = 3) or luminal sodium propionate plus 10 µM HOE-694 in combination with serosal NaCl medium (triangle , n = 4). As shown, either removal of sodium or addition of HOE-694 blocked pHi recovery.

We next questioned whether differences in pHi could be resolved at different regions of the cell along the apical-to-basal axis. Figure 6A shows a SNARF-1 fluorescence image of a cell monolayer directly imaged along the apical-to-basal axis during superfusion with luminal sodium propionate. Superimposed on the image are five regions (each 10 pixels in diameter) that indicate the size and approximate spacing of subcellular domains that were independently analyzed to assess the response along the apical-to-basal axis. Analysis of the five regions was performed at each time point for four or five cells in each monolayer. Figure 6B shows a SNARF-1 ratio image calculated from the cells in Fig. 6A. As shown qualitatively from the pseudocolor scale, the ratio image reports that the apical regions of cells in the monolayer were more acidic than the basal regions. To investigate whether results were influenced by optical artifacts, control experiments questioned whether ratio images of 0.1 mM carboxy SNARF-1-free acid in solution reported constant pH 1) at the interface between the dye solution and a glass coverslip and 2) as a function of focal depth into the dye solution. No pH measurements at the different locations in the dye solution were significantly different (P > 0.7), suggesting that optical artifacts are unlikely to be responsible for pHi differences that are reported at different depths of the cell. These results confirmed our previous results when using a different confocal microscope (6).


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  		Fig. 6.   Direct confocal imaging of polarized HT29-C1 cells along the apical-to-basal axis. Filter-grown HT29-C1 cells were imaged during superfusion with luminal sodium propionate and serosal NaCl medium, at the time point reporting the nadir pHi in whole cell analyses (i.e., time 0 in Fig. 5A). A: confocal fluorescence image of SNARF-1-loaded cells. The xz-plane image is an average of the fluorescence intensities from simultaneously collected 550- to 600-nm and 620- to 680-nm images. Apical and basal boundaries of the cell monolayer are indicated by arrows. Superimposed on the image are 5 circular regions that have the size and placement typically used for subcellular analyses of pHi. B: pseudocolor ratio image derived from data in A. Color bar defines the correspondence of color to pHi. As shown, the apical pole of cells is relatively acidic compared with the basal pole.

Quantitative analysis of subcellular events from a single monolayer is shown in Fig. 7. Figure 7A shows results analyzed for the entire cytosol during a time course experiment compared with results in Fig. 7B, which analyzed five subcellular regions along the apical-to-basal axis (results averaged from 4 cells in the monolayer). As shown, exposure to serosal SCFA caused a prominent acidification of the apical regions of the cell (qualitatively similar to the response to luminal SCFA shown in Fig. 7B) in either the presence or absence of sodium. Results in Fig. 7B also suggest that sodium-dependent pHi recovery occurs prominently in the most apical region of the cell and that pHi gradients can be generated and sustained in the absence of Na/H exchange.


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  		Fig. 7.   Comparison of cellular response to serosal SCFA reported for whole cell and subcellular regions along the apical-to-basal axis. Results are from a time course experiment that imaged a filter-grown HT29-C1 monolayer with a confocal microscope. Cells were directly imaged along the apical-to-basal axis while the response to either serosal sodium propionate (NaProp) or TMA-propionate (TMAProp) was evaluated. A: measurement of pHi reported from whole cell analyses. B: measurement of pHi from 5 subcellular regions spanning the apical-to-basal dimension of the cells. Size and spacing of regions were as depicted in Fig. 6A, and results from each region are reported separately (key identifies most apical, near-apical, mid-cell, near-basal, and most basal from top to bottom, respectively). Subcellular analysis was performed for 5 cells in the monolayer, and average values of pHi are reported.

To substantiate these conclusions, results were compiled from multiple experiments that compared the subcellular pHi values before, during, and after exposure to serosal SCFA. Results in Fig. 8A are from six separate experiments, with five subcellular compartments analyzed in at least four cells for each time point in each experiment. Three time points were examined during serosal SCFA exposure. Time zero is defined as the nadir pHi (as determined from whole cell analyses) directly after SCFA addition. The pHi was also reported after an additional 7 or 14 min of SCFA exposure past time zero. Statistical comparisons (one-way ANOVA with Bonferroni multiple comparison tests) were first made to determine which individual regions significantly changed pHi during the experiment. As predicted from inspection of the results, each cellular region acidified due to serosal sodium propionate addition at time zero (P < 0.05 for each region, comparing before vs. 0 min). During the 14 min of SCFA exposure, only the most apical region underwent significant pHi recovery alkalinization (P < 0.01, comparing 0 vs. 14 min, and P < 0.05, comparing 0 vs. 7 min), although all regions reported increasing values of pHi. All regions alkalinized upon SCFA removal (P < 0.001 for each region comparing 14 min vs. after) and the pHi after SCFA removal was higher than the starting pHi (P < 0.001 for each region, comparing before vs. after). This is consistent with our previous observations of a pHi overshoot following pHi recovery by Na/H exchange during SCFA exposure of isolated mouse colonocytes (8). Statistical comparisons also questioned which regions within the cell were different at any given time point in Fig. 8A. Before SCFA addition, cells were exposed to NaCl medium and were at resting pHi. Under these resting conditions ("before"), the most apical region was significantly acidic compared with the three most basal regions of the cell (P < 0.01), but no other differences were significant among regions. During serosal SCFA exposure, the most apical region was more acidic than all other portions of the cell (P < 0.01), and the adjacent near-apical region was also more acidic than the most basal portion of the cell (P < 0.05). Thus the apical portions of the cells act as a distinct subcellular compartment that is the site of the majority of pHi recovery. After SCFA removal, there was no difference among pH values in intracellular regions.


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  		Fig. 8.   Analysis of subcellular pHi following serosal SCFA addition. Results from confocal time course experiments were compiled to quantitatively compare the subcellular response to serosal sodium propionate in the presence vs. absence of active Na/H exchange. Multiple time points were analyzed. Before SCFA addition ("before"), cells were exposed to NaCl medium in both luminal and serosal superfusates. SCFA medium was then added only to the serosal medium. During SCFA exposure, pHi was analyzed at the nadir pHi determined from whole cell experiments (defined as time 0) and at 7 and 14 min past time 0. When SCFA medium was removed (14-24 min after SCFA addition), it was replaced with NaCl medium and pHi was evaluated 2-4 min later ("after"). Size and spacing of subcellular regions were as depicted in Fig. 6A, and results from each region are reported separately for each time point (key identifies most apical, near-apical, mid-cell, near-basal, and most basal, from top to bottom, respectively). Subcellular analysis was performed for 5 cells in each monolayer at each time point, and average values of pHi are reported. A: cells previously equilibrated in NaCl medium (i.e., at resting pHi) were exposed to serosal 130 mM sodium propionate. Results are compiled from 6 experiments. B: cells were previously exposed to conditions described in A and so were not at resting pHi at the before time point. Cells were exposed to serosal 130 mM sodium propionate plus serosal 10 µM HOE-694 at time 0. Results are compiled from 4 experiments.

In prior experiments, we had shown that transepithelial gradients of SCFAs lead to selective activation of polarized Na/H exchangers. Specifically, serosal propionate preferentially activated basolateral, but not apical, Na/H exchange (46). To determine whether basolateral Na/H exchange was affecting the pHi gradient, similar experiments were performed in the presence of serosal 10 µM HOE-694, to fully inhibit basolateral Na/H exchange. Figure 8B compiles results from multiple experiments (n = 4) that compared subcellular pHi values before, during, and after exposure to serosal SCFA plus 10 µM HOE-694. In this data set, cells had previously been exposed to serosal sodium propionate alone; therefore, the before results in Fig. 8B are similar to the "after" condition in Fig. 8A. In the presence of HOE-694, all cellular regions acidified when SCFA was added and alkalinized when SCFA was removed (P < 0.05), but no pHi recovery trend was noted in any region. Qualitatively similar results were observed when cells were acidified by serosal TMA-propionate to inactivate Na/H exchange (data not shown). In the presence of HOE-694, the most apical region was more acidic than other regions of the cell (P < 0.05) at all time points during SCFA exposure. When Fig. 8A is compared with Fig. 8B, a similar subcellular pHi gradient is noted in both the presence and absence of HOE-694, suggesting that 1) the presence of the SCFA, not the activity of Na/H exchange, was responsible for generating the pHi gradient and 2) the apical acid/base equivalents do not equilibrate with the rest of the cell in the absence of pHi regulation.

Figure 9 presents an analogous compilation of subcellular pHi values following exposure to luminal sodium propionate in either the absence (Fig. 9A; n = 6) or presence (Fig. 9B; n = 3) of 10 µM HOE-694. In response to luminal sodium propionate (Fig. 9A), all regions except the most basal were significantly acidified by SCFA addition and alkalinized after SCFA removal (P < 0.05). Within cells, there was no significant difference among the regional pHi values either before or after SCFA exposure. However, at all time points during SCFA exposure, the most apical region was acidic compared with all other regions (P < 0.05), and the near-apical region was also acidic compared with the most basal region (P < 0.05). Overall, results were qualitatively similar to those following serosal sodium propionate and suggested that the apical portion of the cell was the most responsive site of pHi change elicited by either luminal or serosal SCFA exposure. The results also confirm prior results showing that apical Na/H exchange is predominately activated in response to luminal SCFA (46).


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  		Fig. 9.   Analysis of subcellular pHi following luminal SCFA addition. Results from confocal time course experiments were compiled to quantitatively compare the subcellular response to luminal sodium propionate in the presence vs. absence of active Na/H exchange. Before SCFA addition (before), cells were exposed to NaCl medium in both luminal and serosal superfusates. SCFA medium was then added only to the luminal medium. During SCFA exposure, pHi was analyzed at the nadir pHi determined from whole cell experiments (defined as time 0) and at 7 and 14 min past time 0. When SCFA medium was removed (14-26 min after SCFA addition), it was replaced with NaCl medium and pHi evaluated 2-4 min later (after). Size and spacing of subcellular regions were as depicted in Fig. 6A, and results from each region are reported separately for each time point (key identifies most apical, near-apical, mid-cell, near-basal, and most basal from top to bottom, respectively). Subcellular analysis was performed for 4 or 5 cells in each monolayer at each time point, and average values of pHi are reported. A: cells previously equilibrated in NaCl medium (i.e., at resting pHi) were exposed to luminal 130 mM sodium propionate. Results are compiled from 6 experiments. B: cells were exposed to luminal 130 mM sodium propionate plus luminal 10 µM HOE-694. Results are compiled from 3 experiments.

Comparison of transcellular pHi gradients caused by apical or basolateral SCFA. On the basis of the differential activation of polarized Na/H exchangers by luminal vs. serosal SCFAs (46), we had previously predicted that exposure to luminal SCFA might produce a different subcellular heterogeneity of pHi from exposure to serosal SCFA. To formally test this prediction, we compared the extent of intracellular acidification caused by either luminal or serosal SCFA at each of the five subcellular regions. Results in Fig. 10 were normalized to the pHi change observed at the most basal portion of the cell, to facilitate comparisons between conditions. Both luminal and serosal SCFA produced greater acidification at the apical vs. basolateral pole of the cell (P < 0.001). However, as indicated in Fig. 10, luminal SCFA caused a greater pHi acidification than serosal SCFA in the entire apical half of the cell (P < 0.05).


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  		Fig. 10.   Comparison of transcellular acidification caused by luminal or serosal SCFA. We compared the magnitude of pHi change (Delta pHi) elicited immediately on SCFA addition to either luminal or serosal medium. Delta pHi was quantified for each subcellular region individually as the difference between "0 min" and "before" pHi values from experiments such as those in Figs. 8 and 9. Subcellular heterogeneity of acidification was similar whether caused by sodium propionate in the absence or presence of HOE-694 or from exposure to TMA-propionate (data not shown); therefore, these conditions were compiled together. Values in each monolayer were normalized to the acidification observed at the most basal region of the cell to facilitate transcellular comparisons between conditions. Before normalization, average acidification observed in the most basal region was 0.40 ± 0.05 pH units (13 experiments) when exposed to serosal SCFA and 0.25 ± 0.05 pH units (9 experiments) when exposed to luminal SCFA. * P < 0.05, ** P < 0.01 when luminal vs. serosal responses at the same subcellular site are compared.

Because luminal SCFA predominantly activates apical Na/H exchange (NHE2), and serosal SCFA predominantly activates basolateral Na/H exchange (NHE1) (46), it was also of interest to ask if the pHi recovery in response to these two stimuli was different across the cell. Therefore, we compared the extent of pHi recovery (Na/H exchange) during 14 min of either luminal or serosal SCFA exposure at each of the five subcellular regions. Results are shown in Fig. 11. Both luminal and serosal SCFA elicited a pHi recovery of similar magnitude at the apical region of the cell, which was larger in both cases than that observed at the basal region (P < 0.05). In the basal half of the cell, serosal SCFA elicited a pHi recovery that was consistently greater than that seen during luminal SCFA, but this did not attain statistical significance except for one cell region (indicated in Fig. 11). Qualitatively similar results were observed when results were examined after 7 min of pHi recovery (data not shown). Results suggest that subcellular sites of pHi recovery are similar, although potentially not identical, when comparing between luminal and serosal SCFA exposure.


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  		Fig. 11.   Comparison of transcellular pHi recovery during exposure to luminal or serosal SCFA. We compared the magnitude of pHi change (Delta pHi) elicited during 14 min of sodium propionate exposure at either luminal or serosal surface. Delta pHi was quantified for each subcellular region individually as the difference between "14 min" and 0 min pHi values from experiments such as those in Figs. 8A and 9A. Results are compiled from multiple experiments during exposure to serosal SCFA (6 experiments), or luminal SCFA (6 experiments). ** P = 0.018 when luminal vs. serosal responses at the same subcellular site are compared.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Similar to colonic tissue (17, 19, 20, 37, 39, 40), the colon carcinoma cell line HT29-C1 has both apical and basolateral Na/H exchange activity (46). We previously reported that the polarized Na/H exchangers in HT29-C1 cells could be activated by SCFAs and that flipping the orientation of a transepithelial SCFA gradient preferentially activated either apical or basolateral Na/H exchange (46). Physiologically (luminal-to-serosal) oriented SCFA gradients preferentially activated apical Na/H exchange, in keeping with physiological expectations. These results parallel observations in native tissue (1, 14, 15, 21, 36, 47) and suggest that local activation of Na/H exchangers might be explained by microdomains of pH near the polarized plasma membranes [either within (4) or outside (7) cells]. Our goal in this study was to address two questions raised by these observations. First, we sought to identify the Na/H exchanger isoforms that mediated the response to SCFAs in the HT29-C1 model. Second, we sought to test the prediction that pHi gradients may play a role in selective activation of the polarized exchangers of HT29-C1 cells.

NHE isoforms in HT29-C1 cells. A combination of kinetic and molecular approaches was used to identify which members of the NHE gene family of Na/H exchangers were present in HT29-C1 cells. The Na/H exchange inhibitor HOE-694 is known to have widely different potencies against NHE1 (ID50 = 0.16 µM), NHE2 (ID50 = 5 µM), and NHE3 (ID50 = 650 µM) (12). In suspended or polarized HT29-C1 cells, all Na/H exchange was inhibited by 10 µM HOE-694, suggesting NHE3 was not functionally important. This was consistent with the lack of NHE3 mRNA observed in HT29-C1 cells by RT-PCR. Inhibition kinetics identified two distinct HOE-694-sensitive components of Na/H exchange in suspended HT29-C1 cells, which appeared to segregate into the single kinetic components identified in the apical or basolateral membrane of polarized cells. At a minimum, our work defined the presence of functionally different Na/H exchangers at the two membrane domains of HT29-C1 cells and identified a concentration of HOE-694 (10 µM) that is able to fully inhibit either transporter.

Results allowed us to provisionally assign specific NHE isoforms as the apical and basolateral exchangers of HT29-C1 cells. On the basis of results with HOE-694, the "low-affinity" ID50 value from suspended cells (1.6 µM) was indistinguishable from the value observed at the apical membrane (1.4 µM) and was most closely aligned with known properties of NHE2. In support of the suggestion that NHE2 is the apical Na/H exchanger, NHE2 mRNA was readily detected from HT29-C1 cells. The assignment of the basolateral transporter was less clear because the "high-affinity" ID50 from suspended cells (0.05 µM) was sixfold lower than the ID50 value at the basolateral membrane (0.3 µM). Together, these values bracket the previously reported ID50 value for NHE1 (0.16 µM), and NHE1 mRNA was detected in HT29-C1 cells. The lower affinity at the basolateral membrane could potentially be explained by a mixture of NHE1 and NHE2 in that membrane [NHE2 is a basolateral membrane protein in kidney medulla (49)] or a mixture of NHE1 with another untested isoform having low HOE-694 affinity. However, neither possibility is likely because the basolateral membrane only reports a single HOE-694 binding site and results are not well fit by any mixture of ID50 values in a two-site model (see Fig. 3). Instead, our working hypothesis is that either restricted access or altered environmental conditions (e.g., pH) at the basolateral surface of polarized cells decrease HOE-694 binding to NHE1 and lower the apparent affinity.

Mammalian colon expresses NHE1, NHE2, and NHE3 (18, 52). NHE1 is accepted as a virtually ubiquitous protein, expressed in the basolateral membrane of epithelial cells. It has previously been suggested that 25-55% of NHE1 is expressed in the apical membranes of another HT29 clone (HT29-19A) and in Caco-2 cells (33). Our results suggest a higher fidelity of membrane protein sorting in the HT29-C1 clone under our experimental conditions. NHE2 and NHE3 have been identified as apical exchangers in colonic tissue (2, 18, 26), but it is unclear which isoform(s) contributes to sodium absorption. The current work establishes HT29-C1 as a simplified colonic model system for exploring activation of only a single apical isoform.

Visualizing subcellular pHi gradients. To test for the presence of pHi gradients that may affect activation of NHE1 and NHE2, we applied confocal microscopy to study filter-attached epithelial cells while independently controlling the composition of superfusates bathing the apical and basolateral membrane domains. The focal axis of the microscope was the same as the apical-to-basal axis of the epithelial cells. Given the numerical aperture of the microscope objective (1.2), the refractive index of the water immersion (1.33), and the Stoke's shift of SNARF-1, we should theoretically be able to resolve 0.8 µm along the focal axis using the 488-nm laser for excitation (35). In our work, subcellular measurements reported pHi from 4-µm-diameter regions that were separated by 1-2 µm, well within the capability of our instrument to spatially resolve differences among regions.

There is a concern that the reported subcellular heterogeneity could be explained wholly by optical and/or chemical artifacts. As discussed in RESULTS, measurements of dye in solution under identical conditions have discounted gross optical artifacts (e.g., edge effects, inaccuracy of confocal measurements as a function of focal depth) that could skew results (6). It should also be noted that confocal microscopy faithfully reported pHi recovery in whole cell analyses (Fig. 5), supporting their general validity for measuring pHi. Furthermore, depending on experimental conditions, the disparate pHi results could be reported from multiple cellular regions or alternatively could be completely absent. Thus subcellular pHi was biologically responsive: a property not predicted for an optical artifact.

At the next level, artifact could result from varying chemical properties of SNARF-1 dye at different intracellular regions of the cell. If pHi gradients were purely artifactual, this would require postulating either that SNARF-1 fluorescence is more sensitive to pHi changes at the apical region and/or SNARF-1 has an acid-shifted pKa at the apical regions. However, if a fixed relationship existed between the properties of apical vs. basal dye, then (with the small pH ranges encountered in these experiments) the apical response should always be proportional to the basal response. Contrary to this prediction, results show that the ratio of basal to apical response is not always constant (Figs. 10 and 11). In addition, both postulates would have to be invoked simultaneously to explain how the cell can display transcellular heterogeneity under some, but not all, conditions (Figs. 8 and 9). On the basis of these points, it is unlikely that results are wholly due to subcellular variability in SNARF-1 response, and we therefore assume that subcellular pHi gradients exist and can be regulated by SCFA exposure and Na/H exchange activity. This is consistent with the observation by other investigators of pHi gradients in multiple cell types, as reported using both optical and electrophysiological methods (22, 23, 34, 43).

Generating and regulating transcellular pHi gradients. We know little about the minimum requirements to sustain pHi at distinct values in one subcellular region vs. another. Subcellular heterogeneity is difficult to detect when cells are equilibrated in a conventional NaCl medium (significant in Fig. 9A before but not in Fig. 8A before), so at a minimum there is not yet compelling evidence for the maintenance of subcellular pHi heterogeneity in the absence of SCFA stimuli. In the presence of 130 mM SCFA, transcellular pHi gradients are rapidly established in 1-2 min, in either the absence or presence of active pHi-regulatory mechanisms. Because the apical domain acidifies most in response to either apical or basolateral SCFA, it seems likely that this effect requires predominantly the mere presence of SCFA rather than 1) the vectorial energy in the transepithelial SCFA gradient or 2) a transcellular gradient of SCFA acting as a variable proton buffer. In these experiments, propionate is used as a surrogate for total SCFA concentration (in the 100-150 mM range). This is probably a reasonable assumption, since equimolar amounts of the major SCFAs (C2-C5 aliphatic monocarboxylates) produce similar activation of Na/H exchange in HT29-C1 cells [assayed as amiloride-sensitive swelling (45)]. Furthermore, lower SCFA concentrations are known to produce transient acidification in colonic tissue (14) and isolated colonocytes (8, 16, 47).

During pHi regulation by NHE1 or NHE2, pHi recovery is observed in the apical compartment, implying that protons leave this space (or hydroxyl anions enter it). Results in Fig. 11 suggest that NHE1 does a marginally better job than NHE2 at clearing protons from the basal half of the cell, but there is no other evidence to suggest whether the net proton fluxes that predominate in each region are those that go across the plasma membrane or that mix with other regions.

The simplest explanation for these results would be a gradient of fixed proton buffers in cytosol, with greater buffering capacity in the basal half of the cell (27, 28). This would also require the assumption that proton diffusion is not at equilibrium within cells, which is supported by the observation of pHi gradients by other investigators in other cell types (22, 23, 34, 43). In this model, addition of a bolus of acid or base to the cytosol could produce subcellular changes in pHi proportional to the buffering capacity in each region. This would explain how luminal and serosal SCFA could both cause predominantly subapical pHi acidification and how NHE2 and NHE1 could both cause pHi recovery predominantly in the subapical domains. However, strict proportionality of transcellular pHi changes was not observed when the acidification caused by luminal vs. serosal SCFA was compared (Fig. 10). Therefore, gradients of fixed proton buffers cannot explain all observations. We predicted that transepithelial SCFA gradients would cause polarized changes in pHi (46), and this effect could conceivably have imposed a second force affecting subcellular pHi that was additive with effects of fixed buffer gradients. Further experiments will be needed to test these possibilities.

The apical region was the most interesting from a physiological point of view. It can be defined functionally as a compartment that 1) exists in the apical quarter of the cell, 2) is accessible to SCFAs and/or protons that flux across either apical or basolateral membranes, and 3) is the predominant site of pHi acidification and pHi recovery activated by SCFAs. Some of these properties were correctly predicted by Dahger et al. (14, 15) when examining SCFA and CO2 stimulation of sodium absorption and have been summarized in a recent review (4). We do not have enough information to physically define the apical subcellular compartment. Because adjacent regions in the apical half of the cells can also display significant pHi differences from the basal portions of the cell, there do not appear to be sharp boundaries to the pHi microdomain. This makes it less likely that the apical compartment is a previously unrecognized organelle or a structure associated only with the brush-border membrane. Although SNARF-1 dye will sometimes display punctate fluorescence in subapical areas, above the usual homogeneous cytosolic dye fluorescence, the ratio values reported from these "bright" areas are not different from surrounding areas (data not shown). It should also be noted that because the apical region reports the expected pHi recovery from an acid load, the basal regions may actually be the sites at which unusual behavior is being manifest. In other words, it may be muting of the basal response, rather than amplification of the apical response, that is the essential event in defining unusual transcellular behavior.

Do transcellular pHi gradients explain selective activation of Na/H exchangers by SCFA gradients? Our experiments were designed to compare two transepithelial SCFA gradient conditions that elicited a dramatic alteration in polarized Na/H exchange activation in prior work (46). Based on a model of transepithelial nonionic diffusion, we predicted that luminal SCFA would cause mainly acidification of the apical domains of the cell and serosal SCFA would cause mainly acidification of the basolateral domains. This prediction was only partially fulfilled. In support of the original prediction, luminal SCFA was shown to acidify the apical domain more selectively than serosal SCFA (Fig. 10). However, the model did not predict that both luminal and serosal SCFA would predominantly cause acidification in the apical regions of the cell.

Results affect our working model of SCFA-stimulated Na/H exchange in HT29-C1 cells. The highly reactive apical domain will act as an amplifier of apical NHE2 activity when the cell is acidified. Physiological (apical-to-basolateral) SCFA gradients will also help to enhance selective activation of apical NHE2 by preferential acidification of this subcellular domain adjacent to the membrane. For these reasons, the subcellular response is ideally poised to activate apical exchanger(s) and make sodium absorption exquisitely sensitive to pHi regulation (4, 14, 15). In contrast, the basal cell domains are relatively alkaline and resistant to pHi perturbation, features that should dampen activation of basal NHE1 under all tested conditions. Transcellular pHi results do not explain how serosal SCFA can preferentially activate basolateral NHE1, since this condition still results in striking subapical acidification. With our current resolution of subcellular events, we can only speculate that the activation of NHE1 in the lateral membranes will be intermediate between responses near the apical and basal poles. In summary, pHi can be viewed as an amplifier of cellular Na/H exchange in HT29-C1 cells, with subcellular pHi heterogeneity playing a major role in assuring robust activation of apical NHE2 during physiological stimulation by SCFAs. However, the ability to flip SCFA gradients and predominately activate NHE1 requires that other actions of SCFA gradients [e.g., changes in extracellular pH; (7)] play a dominant role in controlling activation of NHE1 activity and/or inactivation of NHE2. Extrapolation of these conclusions to native tissue must be made with caution, until a similar analysis can be performed to appraise the presence and importance of pHi gradients in the colonic epithelium of animals.


    ACKNOWLEDGEMENTS

Caco-2 mRNA was the generous gift of Dr. C. H. C. Yun. We gratefully acknowledge critical reading of the manuscript by Dr. Chahrzad Montrose-Rafizadeh.


    FOOTNOTES

This work was supported by a Johns Hopkins University Provost's Undergraduate Award to T. Gonda and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42457.

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: M. H. Montrose, Indiana Univ., Med Sci 307, 635 Barnhill Drive, Indianapolis, IN 46202.

Received 26 January 1998; accepted in final form 26 September 1998.


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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(1):G259-G270
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