Three distinct mechanisms of HCO3 secretion in rat distal colon

Sadasivan Vidyasagar, Vazhaikkurichi M. Rajendran, and Henry J. Binder

Department of Internal Medicine, Yale University, New Haven, Connecticut 06520

Submitted 31 October 2003 ; accepted in final form 14 April 2004


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 ABSTRACT
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HCO3 secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO3 secretion have revealed evidence of lumen Cl dependence, suggesting a role for apical membrane Cl/HCO3 exchange, direct examination of HCO3 secretion in isolated crypt from rat distal colon did not identify Cl-dependent HCO3 secretion but did reveal cAMP-induced, Cl-independent HCO3 secretion. Studies were therefore initiated to determine the characteristics of HCO3 secretion in isolated colonic mucosa to identify HCO3 secretion in both surface and crypt cells. HCO3 secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO3 secretion (5.6 ± 0.03 µeq·h–1·cm–2) was abolished by removal of either lumen Cl or bath HCO3; this Cl-dependent HCO3 secretion was also inhibited by 100 µM DIDS (0.5 ± 0.03 µeq·h–1·cm–2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl channel blocker. 8-Bromo-cAMP induced Cl-independent HCO3 secretion (and also inhibited Cl-dependent HCO3 secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl-independent, DIDS-insensitive, saturable HCO3 secretion that was not inhibited by NPPB. Three distinct HCO3 secretory mechanisms were identified: 1) Cl-dependent secretion associated with apical membrane Cl/HCO3 exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO3 exchange.

chloride/bicarbonate exchange; short-chain fatty acid/bicarbonate exchange; anion channel; pH stat


THE MAMMALIAN COLON ABSORBS fluid, Na+, and Cl while secreting K+ and HCO3 (7). Although Na+ and Cl absorption has been studied thoroughly during the past three decades (9), the mechanism of colonic HCO3 secretion has not been investigated as extensively (14, 15, 1720, 25, 38, 52). The generally accepted model of basal HCO3 secretion has included an apical membrane Cl/HCO3 exchange (17, 40). Apical membrane Cl/HCO3 exchanges also have been closely linked to Na+/H+ exchange to account for electroneutral NaCl absorption (40, 42), but they may also function independently of Na+/H+ exchange. Both Cl/HCO3 exchange and Na+/H+ exchange are present in the apical membranes of surface cells of the distal colon, are not present in crypt cells, and are downregulated by cAMP and aldosterone (40–42).

In both clinical diarrhea and experimentally induced in vivo models of diarrhea, a HCO3-rich, plasmalike solution has often been identified (21). The driving force of fluid secretion in these models is active electrogenic Cl secretion, which has been the subject of numerous in vitro investigations (1). In contrast, HCO3 secretion that is induced by one or more secretogogues and second messengers has been studied less frequently and often is not observed in in vitro models. No adequate explanation has been proposed for the failure to observe HCO3 secretion in vitro despite its presence in in vivo models.

Spatial separation of surface cell and crypt cell function has been a long-standing concept of colonic physiology. Absorptive processes are present in surface cells, and secretory processes are present in crypt cells (63). To study crypt cell function directly, we have developed methods for the isolation of 1) individual crypts that allow determination of fluid and electrolyte movement in crypts (25) and 2) apical membrane vesicles (AMV) from relatively pure crypt cell preparations that permit identification of specific transporters in crypt apical membranes (41). Using these experimental approaches, we have studied HCO3 secretion in isolated microperfused crypts from the rat distal colon (25). In these studies, neither endogenous nor Cl-dependent HCO3 secretion was identified, but vasoactive intestinal peptide (VIP), acetyl choline, and dibutyryl cAMP induced HCO3 secretion that required HCO3 in the serosal bath. This HCO3 secretion was not altered by the removal of lumen Cl but was inhibited by lumen 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a nonspecific Cl channel blocker (25). Because these studies were performed in isolated crypts, it is not known whether cAMP-induced HCO3 secretion also occurs in surface cells and, if it does, whether the mechanism of HCO3 secretion in surface cells is similar to or different from that in crypt cells.

Short-chain fatty acids (SCFA) are the major anions in stool but are not present in the diet (8). SCFA are produced by fermentation of nonabsorbed carbohydrate by colonic bacteria. The primary SCFA are acetate, propionate, and butyrate, with butyrate being the primary nutrient for colonocytes (43). In addition to SCFA stimulation of fluid, Na+, and Cl absorption, SCFA also induce HCO3 secretion (62). The model of butyrate stimulation of Na+ and Cl absorption is based on butyrate uptake across the apical membrane via a butyrate/HCO3 exchange (or via nonionic diffusion) linked to apical membrane Na+/H+ and Cl/butyrate exchanges (4, 11, 32, 39). Detailed information regarding the mechanism of SCFA-induced HCO3 secretion is limited.

The present study was initiated to examine HCO3 secretion in isolated intact colonic mucosa with the use of pH stat methods to provide quantitation of HCO3 secretion. The pH stat method with intact colonic mucosa identifies HCO3 secretion that may originate in surface or crypt cells. If the characteristics of HCO3 secretion were found to differ qualitatively from those previously described in isolated crypts, we would conclude that different mechanisms of HCO3 secretion exist in surface and crypt cells. In contrast, if the characteristics of HCO3 secretion in the present studies with the use of pH stat methods were identical to those observed in isolated perfused crypts, it would be uncertain whether HCO3 secretion is present in surface cells as well as in crypt cells. We report the identification, characterization, and interrelationship of three distinct modes of HCO3 secretion: Cl dependent, cAMP stimulated, and SCFA dependent.


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Nonfasting male Sprague-Dawley rats weighing 200–250 g were used in all experiments. Colonic mucosa was obtained from the distal colon after exsanguination, and HCO3 transport studies were performed in mucosa that was stripped of muscular and submucosal layers as described by Fromm et al. (23). Mucosa was mounted between Ussing-type Lucite chambers as previously described (6). All experiments were approved by the Yale University Institutional Animal Care and Use Committee.

pH stat recordings. HCO3 secretion was quantitated with the use of Bi-burette TIM 856 (Radiometer Analytical, Villeurbanne, France), which titrates both above and below stat pH 7.4 with a hysteresis of 0.05 and thus titrates between pH 7.35 and 7.45 (i.e., the physiological limits of pH range for bodily fluids). With the use of this technique, the lumen solution pH was continuously maintained at a constant (or stat) pH by the addition of 0.025 N H2SO4. Pumps were programmed to operate in real time according to the pH changes of the lumen solution, with the pumps calibrated to deliver a minimum of 0.05 µl at a given time. Calibration of standard to stat pH was established by adding increasing concentrations of acid. A known quantity of H2SO4 added to a minimally buffered solution titrated against HCO3 provided a linear curve. The amount of HCO3 measured in the lumen solution was always within the linear range of this curve. The acid used for the titration was diluted in the same ionic solution as that used in that particular experiment to obtain a final concentration of 0.025 M. Colonic tissues were always exposed to a buffered solution on the bath side, while tissues on the lumen side were exposed to a minimally buffered solution (0.1 mM HEPES buffer, pH 7.4) (46). HCO3 secretion is equivalent to the amount of acid required to maintain pH 7.4. All experiments were performed under voltage-clamp conditions, and HCO3-free solutions were gassed with 100% O2, while HCO3-containing solutions were gassed with 95% O2-5% CO2. HCO3 secretion was expressed as microequivalents per hour per square centimeter.

Initial studies demonstrated that immediately after the tissue was mounted, HCO3 secretion was present in the absence of bath HCO3 but rapidly fell toward 0 within 20–30 min. If bath HCO3 was not added, HCO3 secretion remained close to 0. Addition of HCO3 to bath solution resulted in a rapid increase in HCO3 secretion that remained constant for ≥60 min (Fig. 1). All experiments were performed during this 1-h steady-state period.



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Fig. 1. Continuous addition of H2SO4 to lumen solution is required to maintain pH at 7.4 in the presence of lumen Cl and bath HCO3. Cumulative addition of acid over a period of time is represented by the trace. HCO3 secretion was defined as the amount of H2SO4 required to maintain pH at a fixed or stat pH (7.4) and expressed as µeq·h–1·cm–2. HCO3 secretion was linear within 20 min of the addition of bath HCO3 and lasted for ≥60–100 min.

 
In experiments in which inhibitors were added to the mucosal solution, the specific drug was added during the initial steady-state period, pH was adjusted, and tissue was allowed to equilibrate for 30 min until a steady rate of HCO3 secretion was again observed. In experiments in which the inhibitor was added to the serosal side, the tissue was also equilibrated for 30 min to achieve a steady state of HCO3 secretion. In experiments in which isobutyrate was used, 25 mM isobutyrate solution was first neutralized with N-methyl-D-glucamine (NMDG) before use. The composition of the several solutions used in these experiments is presented in Table 1. In brief, in Cl-free experiments, isethionate was used as a substitute for Cl; in Na+-free experiments, NMDG was used as a Na+ substitute. Glucose (10 mM) was added to all solutions. Only one tissue from each animal was used for a specific experiment, and only one experimental condition was used with each tissue. All experiments were repeated at least four times.


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Table 1. Composition of several solutions

 
Ion flux studies. Unidirectional Na+ and Cl fluxes were performed across isolated colonic mucosa with 22Na and 36Cl under short-circuit conditions (DVC 1000; World Precision Instruments, Sarasota, FL) as previously described (3, 6). Tissues were paired on the basis of a conductance difference of <10%. Net ion fluxes (Jnet) were then calculated from the difference between mucosal-to-serosal transport (Jms) and serosal-to-mucosal transport (Jsm). Positive values reflect net absorption, and negative values reflect net secretion. Samples were collected from the cold side after a 15-min equilibration period and thereafter at 15-min intervals for three time periods.

Statistics. Results are presented as means ± SE. Statistical analyses were performed with the use of paired and unpaired t-tests and Bonferroni's one-way ANOVA post hoc test. P < 0.05 was considered significant.

Chemicals and solutions. Amiloride, 8-bromo-cAMP, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), dimethyl sulfoxide grade I, glibenclamide, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), isethionic acid, NMDG, NPPB, potassium gluconate, and sodium gluconate were obtained from Sigma. 36Cl and 22Na were obtained from Amersham Biosciences (Piscataway, NJ).


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Cl-dependent HCO3 secretion. The initial experiment was performed to determine whether there was endogenous secretion of HCO3, i.e., in the presence of a nominally HCO3-free solution in the serosal or bath solution of rat distal colon. Under conditions in which the colonic mucosa was bathed on both the lumen and bath sides with a nominally HCO3-free Ringer solution, only a minimal rate of HCO3 secretion was observed (0.3 ± 0.03 µeq·h–1·cm–2) in the presence of lumen Cl.

The addition of HCO3 to the bath solution resulted in a substantial rate of HCO3 secretion (5.7 ± 0.04 µeq·h–1·cm–2), indicating that basal HCO3 secretion required bath HCO3 (Fig. 2). Because the methodology of these pH stat studies requires an unbuffered or minimally buffered lumen solution (i.e., one that is nominally HCO3-free), experiments could not be performed to assess whether the presence of HCO3 in the lumen solution modified HCO3 secretion. To establish that the observed rate of HCO3 secretion was not masking the actual rate of basal secretion as a result of its partial neutralization by simultaneous proton secretion, e.g., K+/H+ exchange, experiments were performed in which potential proton secretion into the lumen solution was inhibited. Table 2 demonstrates that in the presence of 1 mM orthovanadate, an inhibitor of P-type ATPases, HCO3 secretion was significantly increased by ~7%, suggesting that HCO3 secretion was only minimally reduced by parallel K+-dependent proton secretion (i.e., H+-K+-ATPase). HCO3 secretion was also measured in experiments in which apical membrane Na+/H+ exchange was inhibited by the removal of lumen Na+, by the addition of 1 mM amiloride to the lumen solution, and by both the removal of lumen Na+ and the addition of amiloride. Inhibition of Na+/H+ exchange in each of these three experimental conditions resulted in a minimal though significant decrease in the rate of HCO3 secretion (Table 2). As a result, the observed HCO3 secretion represented net HCO3 secretion.



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Fig. 2. Role of bath HCO3 on HCO3 secretion. HCO3 (25 mM) was replaced by 25 mM isethionate. Cl-dependent HCO3 secretion with 119.8 mM Cl present in lumen solution is shown. Numbers in parentheses indicate number of tissues studied in each group. *P < 0.001 compared with group without bath HCO3.

 

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Table 2. Effect of apical membrane Na+/H+ exchange and H+-K+-ATPase inhibition on Cl-dependent HCO3 secretion

 
To determine whether basal HCO3 secretion was Cl dependent, we measured HCO3 secretion in the absence of lumen Cl. As shown in Fig. 3, the absence of Cl from the lumen solution resulted in almost complete elimination of basal HCO3 secretion (0.6 ± 0.02 µeq·h–1·cm–2), indicating that basal HCO3 secretion was lumen Cl dependent. Thus basal HCO3 secretion is completely dependent on lumen Cl and as a result is referred to as Cl-dependent HCO3 secretion.



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Fig. 3. Effect of anion transport inhibitors on Cl-dependent HCO3 secretion. Open bar represents the group without lumen Cl; hatched bar represents lumen Cl group without any transport inhibitors present. Cl-dependent HCO3 secretion also was measured in the presence of 3 transport inhibitors (crosshatched bars): 100 µM DIDS, 100 µM acetazolamide (ACZ), and 100 µM 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB). Numbers in parentheses indicate number of tissues studied in each group. *P < 0.001 compared with group without lumen Cl. **P < 0.001 compared with lumen Cl group without any transport inhibitors. +Not significantly different from lumen Cl group without any transport inhibitors.

 
To establish whether Cl-dependent HCO3 secretion involves an apical membrane Cl/HCO3 exchange, we performed experiments with an anion exchange inhibitor, DIDS (100 µM). DIDS, which at 100 µM concentration is an inhibitor of Cl/anion exchanges (40, 57), completely inhibited HCO3 secretion (0.5 ± 0.04 µeq·h–1·cm–2), which is consistent with a Cl/HCO3 exchange being closely associated with HCO3 movement across the apical membrane (Fig. 3). Experiments were also performed to assess the effect of acetazolamide, a carbonic anhydrase (CA) inhibitor, on Cl-dependent HCO3 secretion. Acetazolamide (100 µM) substantially reduced Cl-dependent HCO3 secretion (Fig. 3). Further experiments were performed to determine whether an apical membrane Cl/HCO3 exchange is a carrier-mediated process, by assessing its kinetic characteristics. Increasing concentrations of lumen Cl resulted in an enhanced but saturable rate of HCO3 secretion with evidence of saturation kinetics (Fig. 4). These experiments yielded a Km for Cl of 1.0 ± 0.03 mM and a Vmax of 5.6 ± 0.03 µeq·h–1·cm–2.



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Fig. 4. Effect of increasing lumen Cl concentration on Cl-dependent HCO3 secretion. Nonlinear curve fit with the Michaelis-Menten model for enzyme kinetics resulted in Vmax = 5.6 ± 0.03 µeq·h–1·cm–2 and Km = 1.0 ± 0.03 mM. These results were based on experiments in 4 tissues.

 
Because VIP-stimulated HCO3 secretion in studies performed with isolated colonic crypts was inhibited by NPPB, a nonspecific Cl channel blocker (24, 57), the effect of NPPB on Cl-dependent HCO3 secretion was also determined. NPPB (100 µM) did not alter Cl-dependent HCO3 secretion (Fig. 3). Because NPPB inhibits Cl (as well as other anion) channel function, but not Cl/anion exchanges, the failure of NPPB to alter Cl-dependent HCO3 secretion is consistent with the close association of an apical membrane Cl/HCO3 exchange process with Cl-dependent HCO3 secretion that is not a result of the coupling of a Cl/HCO3 exchange with an apical membrane Cl channel.

The observation that the removal of lumen Na+ or the addition of amiloride (Table 2) did not increase the rate of Cl-dependent HCO3 secretion suggests that in the conditions required to perform these pH stat studies (i.e., the presence of a bath-to-lumen HCO3 gradient), apical membrane Na+/H+ exchange is either absent or not coupled to Cl/HCO3 exchange. To establish Na+/H+ exchange function in the presence of a bath-to-lumen HCO3 gradient, we performed Na+ and Cl isotopic flux studies in the presence of HCO3-containing Ringer solution in the bath side and HCO3-free, minimally buffered solution in the lumen side. HCO3-containing solution was bubbled with CO2, and HCO3-free solution was bubbled with O2. Table 3 shows data demonstrating the absence of net Na+ absorption (–1.0 ± 0.4 µeq·h–1·cm–2) and net Cl absorption (5.1 ± 0.2 µeq·h–1·cm–2). Of interest is that the rate of Cl-dependent HCO3 secretion is approximately equal to that of net Cl absorption and is consistent with the presence of a Cl/HCO3 exchange.


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Table 3. Unidirectional and net Na+ and Cl fluxes

 
cAMP-stimulated HCO3 secretion. Increases in mucosal cAMP have multiple effects on colonic ion transport, including stimulation of HCO3 secretion in experiments with isolated crypts (25) and downregulation of apical membrane Cl/HCO3 exchange (3). As a result, it was possible that cAMP might stimulate HCO3 secretion and/or inhibit Cl-dependent HCO3 secretion in the present studies with isolated intact colonic mucosa that included both surface and crypt cells. Therefore, the effect of 8-bromo-cAMP on HCO3 secretion was characterized. As shown in Fig. 5, in the absence of lumen Cl, HCO3 secretion was minimal, but the addition of 0.5 mM 8-bromo-cAMP resulted in a marked increase in HCO3 secretion. To determine whether cAMP-stimulated HCO3 secretion required bath HCO3, as was demonstrated for Cl-dependent HCO3 secretion (Fig. 2), the rate of HCO3 secretion in the presence of 8-bromo-cAMP was established in the absence of bath HCO3. Figure 5 demonstrates that cAMP-stimulated HCO3 secretion was markedly inhibited by the removal of bath HCO3.



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Fig. 5. Effect of cAMP and bath HCO3 on Cl-independent HCO3 secretion. HCO3 secretion was determined in the absence or presence of 8-bromo-cAMP and/or bath HCO3 as indicated. Numbers in parentheses indicate number of tissues studied in each group. *P < 0.001 compared with group without cAMP. **P < 0.001 compared with cAMP, bath HCO3 group.

 
To assess whether cAMP-stimulated HCO3 secretion was associated with an apical membrane anion channel, we also examined the effect of NPPB, a nonspecific Cl channel blocker. Figure 6 demonstrates that HCO3 secretion in the presence of 8-bromo-cAMP was completely inhibited by 100 µM NPPB (0.5 ± 0.03 µeq·h–1·cm–2), indicating that cAMP mediates HCO3 secretion via apical membrane anion channels. Because NPPB at 100 µM concentration is a nonspecific Cl channel blocker (24, 57), studies were also performed with 300 µM glibenclamide, a concentration at which glibenclamide is a relatively specific inhibitor of CFTR, and with 1 mM DIDS, a concentration at which DIDS inhibits the outwardly directed Cl conductance. Figure 6 demonstrates that cAMP-stimulated HCO3 secretion was completely inhibited by 300 µM glibenclamide but was not altered by 1 mM DIDS (4.9 ± 0.08 µeq·h–1·cm–2), suggesting a role for CFTR in cAMP-induced HCO3 secretion. To exclude an effect of glibenclamide on apical membrane K+ channels, the addition of 5 mM barium to the luminal solution was evaluated, but it did not affect HCO3 secretion (data not shown).



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Fig. 6. Effect of anion channel blockers on cAMP-induced HCO3 secretion in the absence of lumen Cl. HCO3 secretion was measured in presence of 8-bromo-cAMP (hatched bar; control). cAMP-stimulated HCO3 secretion was also measured in the presence of 3 channel blockers (crosshatched bars): 100 µM NPPB, 300 µM glibenclamide (Glib), and 1 mM DIDS. Numbers in parentheses indicate number of tissues studied in each group. *Not significantly different from control group; **P < 0.001 compared with control group.

 
Parallel studies performed in the presence of lumen Cl provided an opportunity to determine whether cAMP also modifies Cl-dependent HCO3 secretion. cAMP-stimulated HCO3 secretion was not altered by the removal of lumen Cl (4.9 ± 0.09 vs. 5.1 ± 0.01 µeq·h–1·cm–2), indicating that cAMP-stimulated HCO3 secretion was Cl independent and suggesting that cAMP inhibited Cl-dependent HCO3 secretion. Additional experiments were performed with anion transport inhibitors to confirm that cAMP modified Cl-dependent HCO3 secretion. Figure 7 demonstrates that NPPB inhibited HCO3 secretion to a rate identical to that observed in the absence of lumen Cl (0.5 ± 0.03 vs. 0.4 ± 0.04 µeq·h–1·cm–2). In contrast, 100 µM DIDS did not inhibit cAMP-stimulated HCO3 secretion. Although acetazolamide markedly inhibited Cl-dependent HCO3 secretion (Fig. 3), cAMP-induced HCO3 secretion in the presence of lumen Cl was only minimally reduced by acetazolamide (Fig. 7). These results provide compelling evidence that Cl-dependent HCO3 secretion is absent in the presence of cAMP. Thus cAMP both induced Cl-independent HCO3 secretion via an anion channel and inhibited Cl-dependent HCO3 secretion.



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Fig. 7. Effect of anion transport inhibitors on HCO3 secretion in the presence of lumen Cl and 8-bromo-cAMP. 8-bromo-cAMP-stimulated HCO3 secretion was measured in the presence of lumen Cl (hatched bar; control). Anion transport inhibitors were used to characterize cAMP-stimulated HCO3 secretion (crosshatched bars): 100 µM NPPB, 1 mM DIDS, and 100 µM ACZ. Numbers in parentheses indicate number of tissues studied in each group. *P < 0.001 compared with control group. **Not significantly different from control group. +P < 0.01 compared with control group.

 
SCFA-dependent HCO3 secretion. In vivo studies of HCO3 secretion previously demonstrated that SCFA increases HCO3 secretion but did not establish the mechanism of its effects (62). To assess the effect of the transport of a SCFA rather than its metabolism, we performed these SCFA studies with isobutyrate, a poorly metabolized SCFA. In another series of studies, 25 mM isobutyrate was added to a luminal Cl-free solution, and both the rate and the characteristics of HCO3 secretion were determined. The addition of 25 mM isobutyrate significantly enhanced HCO3 secretion to 5.7 ± 0.05 µeq·h–1·cm–2 (Table 4), a rate that is identical to that of Cl-dependent HCO3 secretion (Fig. 3). Isobutyrate-dependent HCO3 secretion was also determined in the absence of bath HCO3. Isobutyrate-dependent HCO3 secretion was substantially reduced by the removal of bath HCO3 (0.5 ± 0.03 vs. 5.6 ± 0.03 µeq·h–1·cm–2), and this rate was similar to the rate of Cl-dependent HCO3 secretion in the absence of bath HCO3. These observations exclude the possibility that the observed luminal alkalinization that we proposed represented HCO3 secretion was due to either H+-SCFA cotransport or nonionic diffusion.


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Table 4. Effect of isobutyrate on Cl-dependent HCO3 secretion

 
Because the rate of isobutyrate-dependent HCO3 secretion (in the absence of lumen Cl) was similar to that of Cl-dependent HCO3 secretion (Table 4), HCO3 secretion was determined in the presence of both lumen isobutyrate and lumen Cl to determine whether HCO3 secretion that was mediated by lumen isobutyrate and lumen Cl represented the same or different HCO3 secretory processes. HCO3 secretion in the presence of both lumen isobutyrate and lumen Cl was no greater than the rate of HCO3 secretion observed in the presence of either lumen isobutyrate or lumen Cl alone. To establish whether isobutyrate inhibited Cl-dependent HCO3 secretion or whether isobutyrate-dependent HCO3 secretion was not manifest in the presence of Cl, we compared the effect of anion transport inhibitors on HCO3 secretion in the presence of both lumen isobutyrate and lumen Cl with that observed in the presence of lumen Cl alone. Although DIDS and acetazolamide inhibited Cl-dependent HCO3 secretion (Fig. 3 and Table 4), HCO3 secretion in the presence of both isobutyrate and lumen Cl was not substantially altered by either DIDS or acetazolamide. To exclude the possibility that isobutyrate induced HCO3 secretion that was mediated by anion channels, we also examined the effect of NPPB. NPPB (100 µM) did not affect HCO3 secretion in the presence of isobutyrate and lumen Cl. These results establish that in the presence of both isobutyrate and Cl, HCO3 secretion is insensitive to DIDS, acetazolamide, and NPPB, and thus isobutyrate both stimulated Cl-independent HCO3 secretion and inhibited Cl-dependent HCO3 secretion.

To characterize further SCFA-dependent HCO3 secretion, we performed kinetic experiments. Figure 8 demonstrates that increasing concentrations of isobutyrate in the absence of lumen Cl resulted in an enhanced rate of HCO3 secretion with evidence of saturation kinetics with a kinetic constant Km for isobutyrate of 2.9 ± 0.14 mM and a Vmax of 5.9 ± 0.08 µeq·h–1·cm–2. These results permit the conclusion that isobutyrate-dependent HCO3 secretion is mediated by an apical membrane NPPB-insensitive, DIDS-insensitive, Cl-independent, carrier-mediated transport mechanism. Thus isobutyrate-dependent HCO3 secretion is likely secondary to apical membrane SCFA/HCO3 exchange.



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Fig. 8. Effect of increasing luminal isobutyrate concentration on HCO3 secretion. Nonlinear curve fit using the Michaelis-Menten model for enzyme kinetics resulted in Vmax = 5.7 ± 0.03 µeq·h–1·cm–2 and Km = 2.9 ± 0.14 mM. These results were based on experiments in 4 tissues.

 
Because the data presented in Table 4 indicate that Cl-dependent HCO3 secretion was inhibited by isobutyrate, a final series of experiments were designed to assess the effect of isobutyrate on cAMP-stimulated HCO3 secretion. Figure 6 shows that in the absence of isobutyrate, cAMP-induced HCO3 secretion was NPPB sensitive. When isobutyrate was present together with cAMP, HCO3 secretion was not altered by NPPB (5.7 ± 0.05 vs. 5.7 ± 0.07 µeq·h–1·cm–2). Because these experiments were performed in the absence of lumen Cl, HCO3 secretion was Cl independent and NPPB insensitive. These characteristics are those of SCFA-dependent HCO3 secretion and not those of cAMP-stimulated HCO3 secretion, suggesting that isobutyrate also inhibited cAMP-stimulated HCO3 secretion.


    DISCUSSION
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 ABSTRACT
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HCO3 secretion is an important component of normal colonic fluid and electrolyte movement and is a major fraction of the fluid secreted in several diarrheal diseases (21). Despite the essential role of HCO3 secretion both in health and in diarrhea, the mechanism of HCO3 movement in general and HCO3 secretion in particular in both the small and large intestines has been studied relatively infrequently, especially compared with the extensive experimental studies of Cl secretion. Several factors have probably contributed to this relative understudy of colonic HCO3 transport. 1) There are technical difficulties involved in the determination of HCO3, e.g., the inability to use isotopes to measure HCO3 flux at pH 7.4. 2) HCO3 secretion has often been studied in in vitro studies by determining so-called residual flux (Jr), i.e., the portion of the short-circuit current that is not accounted for by measured Na+, Cl, and K+ fluxes (18). The use of Jr to determine HCO3 movement is indirect and is subject to significant experimental variability. 3) HCO3 secretion, when estimated from Jr, can be highly variable in in vitro experiments and is often substantially less than that observed in in vivo studies. An adequate explanation for the lower rates of HCO3 secretion observed in in vitro studies compared with those observed in in vivo studies has not been identified.

Several investigators have used an alternate experimental approach, pH stat, to determine HCO3 movement in vitro in several intestinal epithelia (13, 19, 20, 26, 33, 45, 46, 48, 52). In this method, low concentrations of acid are added to maintain the mucosal solution at a fixed pH (i.e., 7.4). HCO3 secretion is assumed to be equivalent to the amount of acid added to maintain the designated stat pH. This general approach has been used to determine HCO3 movement in gastric, duodenal, ileal, and colonic mucosa (13, 19, 20, 26, 33, 45, 46, 48, 52, 56). We adapted this methodology to study HCO3 secretion in the rat distal colon to complement prior studies of HCO3 secretion in isolated colonic crypt glands (25). HCO3 secretion determined with this approach could theoretically be underestimated if there were also simultaneous proton secretion. In the present experiments, proton secretion resulted in <10% underestimation of HCO3 secretion, as inhibition of H+-K+-ATPase resulted in only a minimal but significant increase in HCO3 secretion (Table 2).

The model of NaCl absorption in the rat distal colon that has been developed during the past 20 years is the presence in the apical membrane of parallel ion exchangers, Na+/H+ exchange and Cl/HCO3 exchange, coupled with intracellular pH (pHi) (7). The relationship between Cl/HCO3 exchange and Na+/H+ exchange in the rat distal colon is complex. Ion fluxes performed under voltage-clamp conditions in the absence of ion gradients have generally revealed approximately equal rates of net Na+ and net Cl absorption (3, 4, 7), and inhibition of Na+/H+ exchange by 1 mM amiloride decreases net Cl absorption (3, 35). Coupling of these two ion exchanges has been demonstrated in studies with brush-border vesicles prepared from rabbit ileum as well as in luminal perfusion experiments in human ileum and rat colon (30, 37, 60). Nonetheless, both exchanges are functionally independent of Cl and Na+, because ion uptake studies with the use of AMV prepared from rat distal colon have demonstrated Na+/H+ exchange and Cl/HCO3 exchange activities that are independent of Cl and Na+, respectively (40, 42). Furthermore, in the proximal small intestine, HCO3 absorption is a result of an apical membrane Na+/H+ exchange that is present in the absence of an anion exchanger (61), while in the colon, when the rate of Cl/HCO3 exchange is greater than that of Na+/H+ exchange, both Cl-dependent HCO3 secretion and net NaCl absorption have been observed (17).

The present studies demonstrate Cl-dependent HCO3 secretion with the use of pH stat methodology that requires that either no buffer or a very low buffering capacity be present in the lumen solution. Table 2 demonstrates that the removal of lumen Na+ and/or the addition of 1 mM amiloride to the lumen solution did not increase HCO3 secretion. The failure to observe an enhancement in HCO3 secretion after inhibition of apical membrane Na+/H+ exchange may be a result of the fact that in the presence of bath-to-lumen HCO3 gradient, an apical membrane Na+/H+ exchange is absent. To address this possibility, we measured Na+ and Cl isotopic flux under the identical experimental conditions that were used in the HCO3 secretion studies, i.e., the presence of a bath-to-lumen HCO3 gradient (Table 3). These studies demonstrated an absence of net Na+ absorption and a rate of net Cl absorption that was approximately equal to that of HCO3 secretion determined in the pH stat studies (5.1 ± 0.2 vs. 5.7 ± 0.04 µeq·h–1·cm–2). The rate of net Cl absorption in the present experiments is approximately equal to that observed in several prior studies of Cl movement across the rat distal colon (3, 4, 7, 58). These experiments provide compelling evidence that the presence of a HCO3 gradient results in inhibition of net Na+ absorption and explains the failure to observe an increase in Cl-dependent HCO3 secretion after experimental maneuvers to inhibit apical membrane Na+/H+ exchange (Table 3). One possible explanation for this observation is that HCO3 movement from bath to cell (in the absence of adequate luminal buffering) raises pHi, thus reducing the driving force for Na+/H+ exchange. Future studies of pHi in surface cells of rat distal colon are required to explore these relationships.

Investigation of colonic HCO3 secretion has been undertaken in several species, including humans, during the past 50 years (14, 15, 17, 19, 20, 25, 38, 52). Most of these in vivo studies have used a luminal perfusion methodology and have largely made similar observations: HCO3 secretion appears to be an active transport process as luminal concentrations of HCO3 increase above plasma concentrations, and the electrical potential difference is lumen negative (14, 15, 17, 38). In general, HCO3 secretion has been established as lumen Cl dependent and is attributed to an apical membrane Cl/HCO3 exchange. Evidence in support of an apical membrane Cl/HCO3 exchange as the mechanism of colonic HCO3 secretion has also been provided by observations in congenital chloridorrhea, a diarrheal disorder associated with high stool Cl concentrations (27, 36). Luminal perfusion studies in these patients established the absence of Cl-dependent HCO3 exchange in the ileum and the colon (but not in the jejunum) (2, 59), and mutations in the DRA (downregulated in adenoma) gene have been identified as the cause of congenital chloridorrhea (34, 36). Furthermore, DRA has been identified as an anion transporter (47). Studies in normal colon have also established the presence of HCO3-dependent Cl absorption (4). Although Cl/HCO3 exchange is coupled to Na+/H+ exchange, resulting in electroneutral NaCl absorption, overall net HCO3 secretion is observed when the rate of Cl/HCO3 exchange is greater than that of Na+/H+ exchange (17).

Several specific models of HCO3 secretion have been identified in different epithelia (13, 17, 28, 29, 49, 53, 55). The source of HCO3 can be either endogenous synthesis from CO2 and H2O by enzymatic action of CA or bath HCO3 with transport across the basolateral membrane. In human and bovine airway cells, HCO3 secretion is linked to endogenous CO2 production with HCO3 movement across the apical membrane and proton movement across the basolateral membrane via Na/H exchange (49). HCO3 secretion that is linked to endogenous CO2 production has been reduced by inhibition of CA (38). In addition to Cl/HCO3 exchange, additional mechanisms proposed for the movement of HCO3 across the intestinal apical membrane include 1) CFTR with a finite HCO3 conductance, 2) a non-CFTR HCO3 channel, and 3) CFTR coupled to Cl/HCO3 exchange (13, 26, 29, 53, 54).

Recent studies have suggested a close association between CA-II and apical membrane Cl/anion exchangers (i.e., AE1, DRA) (50, 51) and may explain a portion of acetazolamide inhibition of HCO3 secretion. Although acetazolamide does not have an effect on Cl/HCO3 exchange in membrane vesicle systems (32), recent studies have indicated that acetazolamide inhibits Cl/HCO3 exchange to a greater degree than that which can be attributed to CA inhibition, suggesting that CA and apical membrane Cl/HCO3 exchange are linked (50, 51). Although Cl-dependent HCO3 secretion was inhibited by acetazolamide (Fig. 3), acetazolamide did not inhibit or only minimally inhibited cAMP-induced and SCFA-dependent HCO3 secretion, respectively (Fig. 7 and Table 4), which indicates that apical membrane anion channel and SCFA/HCO3 exchange are not coupled to CA-II. Although the molecular identity of SCFA/HCO3 exchange is not known, these results permit the speculation that neither AE1 nor DRA encodes the SCFA/HCO3 exchange protein.

The paradigm of colonic ion function is presently a separation of crypt cell and surface cell function (63). Absorptive processes are largely confined to surface cells, while secretory processes are localized in crypt cells. HCO3 secretion has been studied successfully in isolated colonic crypts, and those studies failed to identify basal or Cl-dependent HCO3 secretion (25). This observation is consistent with the localization of Cl/HCO3 exchange to apical membranes of surface cells but not to those of crypt cells (40). Furthermore, the finding of Cl-independent HCO3 secretion in the crypt contrasts with in vitro experiments with intact mucosa in which HCO3 secretion was identified as Cl dependent. These observations indicate that Cl-dependent HCO3 secretion is a surface cell function, while Cl-independent secretion is most likely a crypt cell function.

cAMP stimulated HCO3 secretion both in isolated crypts (25) and in the present studies with intact mucosa that included both surface and crypt cells. HCO3 secretion as determined in these pH stat studies could represent HCO3 that was secreted from crypt and/or surface cells. Because cAMP-stimulated Cl secretion is most likely predominantly restricted to crypt cells (1), cAMP-stimulated HCO3 secretion might also be an exclusive crypt cell function. However, it is not known whether HCO3 secretion is also generated from surface cells, because intact mucosa containing both surface and crypt cells was used in the present studies. Such a conclusion requires the study of surface cells independently of crypt cells.

Besides the induction of HCO3 secretion in colonic crypts and possibly in colonic surface cells, cAMP has several other effects on colonic ion transport (1, 3, 22). cAMP stimulates both active Cl secretion and active K+ secretion (1, 22). Electroneutral NaCl absorption is also inhibited by increased intramucosal concentrations of cAMP (3); the reduction of electroneutral NaCl absorption is most likely a result of its inhibition of Na+/H+ exchange and/or Cl/HCO3 exchange. In the present studies, cAMP not only stimulated HCO3 secretion but also was associated with downregulation of Cl-dependent HCO3 secretion, which most likely was a result of cAMP inhibition of apical membrane Cl/HCO3 exchange (Fig. 7). Although in intact mucosa cAMP appears to completely inhibit apical DIDS-sensitive, Cl-dependent HCO3 secretion, cAMP has not been reported to inhibit the Cl/HCO3 exchange activity of either DIDS-sensitive AE1 or relatively DIDS-insensitive DRA when they are expressed in the in vitro recombinant expression system (12). The possibility that cAMP-stimulated HCO3 secretion represented the coupling of an apical membrane Cl/HCO3 exchange to CFTR was also excluded by demonstrating that the removal of lumen Cl did not affect cAMP-induced HCO3 secretion in either the absence (3.4 ± 0.05 vs. 3.6 ± 0.09 µeq·h–1·cm–2) or presence (4.8 ± 0.02 vs. 5.2 ± µeq·h–1·cm–2) of bath Cl. This indicates that cAMP-stimulated HCO3 secretion was a result of an apical membrane anion channel and was not due to the coupling of a Cl/HCO3 exchange to an apical membrane anion channel (Figs. 6 and 7).

The mechanism of the uptake of SCFA across the apical membrane of colonocytes has been controversial, with evidence of both an apical membrane butyrate/HCO3 exchange and nonionic diffusion (11, 32). It is not unlikely that both mechanisms are operative, with their relative contribution to overall SCFA uptake varying as a function of species and one or more experimental conditions. Several in vivo studies that have observed HCO3 secretion in association with butyrate absorption support the possibility that butyrate uptake across the apical membrane occurs via a SCFA/HCO3 exchange. Table 4 demonstrates that isobutyrate stimulates Cl-independent HCO3 secretion that is insensitive to both DIDS and NPPB. The presence of saturation kinetics (Fig. 8) provides compelling support for a carrier-mediated exchange process that is responsible for butyrate absorption and HCO3 secretion. The failure of DIDS to inhibit butyrate-dependent HCO3 secretion parallels prior observations of apical membrane vesicles in which butyrate/HCO3 exchange was also DIDS insensitive (32).

SCFA-dependent HCO3 secretion potentially could be secondary either to an apical membrane SCFA/HCO3 exchange or to SCFA-mediated proton uptake across the apical membrane. The available evidence supports a critical role for an anion exchange in isobutyrate-dependent HCO3 secretion. First, prior studies of [14C]butyrate uptake by AMV prepared from rat distal colon established the presence of a DIDS-insensitive butyrate/HCO3 exchange (32). Second, in these studies with AMV, an outward-directed pH gradient in the absence of HCO3/CO2 resulted in only minimal butyrate uptake (32), an observation inconsistent with the presence of a H+-SCFA cotransport mechanism. Third, nonionic diffusion of SCFA across the apical membrane could also result in intracellular acidification, but such a process would not result in the saturation kinetics shown in Fig. 8. In addition, there was only minimal butyrate uptake across AMV via nonionic diffusion in the AMV studies (32). Fourth, in the pH stat studies of colonic HCO3 secretion, removal of bath HCO3/CO2 completely inhibited SCFA-dependent HCO3 secretion (Fig. 3), which provides additional evidence against the possibility that SCFA-H+ cotransport across the apical membrane is responsible for isobutyrate-dependent HCO3 secretion in the present studies.

Similarly to cAMP, butyrate modifies several different parameters of colonic ion transport and manifests some specific interactions with cAMP (4, 5, 10, 16, 31). First, butyrate enhances electroneutral NaCl absorption (4). Second, butyrate downregulates both cAMP- and cGMP-induced active Cl secretion but does not modify intracellular Ca2+-mediated Cl secretion (10, 16). Third, although stimulation of butyrate-dependent NaCl absorption is closely linked to Na+/H+ exchange, cAMP does not inhibit butyrate-stimulated NaCl absorption (5). There is evidence that SCFA-dependent electroneutral NaCl absorption is not altered by cAMP as a result of cAMP activation of the NHE2 isoform, which mediates butyrate-dependent, but not HCO3-dependent, Na+ absorption (31).

The present studies identified additional effects of butyrate on colonic ion transport as butyrate blocked both cAMP-induced HCO3 secretion and Cl-dependent HCO3 secretion (Table 4). Evidence that isobutyrate inhibited cAMP-induced HCO3 secretion was provided by the demonstration that HCO3 secretion was NPPB sensitive in the absence of isobutyrate (0.4 ± 0.004 µeq·h–1·cm–2) but was NPPB insensitive in its presence (5.7 ± 0.07 µeq·h–1·cm–2). In addition, isobutyrate also downregulates Cl-dependent HCO3 secretion, based on the observation that HCO3 secretion in the absence of isobutyrate is Cl dependent and DIDS sensitive (Fig. 7). After the addition of isobutyrate, HCO3 secretion is both Cl independent and DIDS insensitive. Thus it appears that SCFA downregulates HCO3 secretion that is regulated either by cAMP-induced anion channels or by Cl/anion exchanges. The mechanisms by which SCFA downregulates Cl-dependent and cAMP-induced HCO3 secretion have not been established. However, SCFA uptake across the apical membrane, via either SCFA/HCO3 exchange or SCFA-mediated proton influx, would undoubtedly lower pHi, thus resulting in a decrease in Cl-dependent and/or cAMP-induced HCO3 secretion. Delineation of these events requires additional studies, including studies of pHi regulation by SCFA.

In conclusion, the present studies demonstrate the presence of three distinct HCO3 transport processes: Cl-dependent, SCFA-dependent, and cAMP-stimulated HCO3 secretion (Fig. 9). There is a substantial interrelationship between these three HCO3 transport mechanisms: cAMP downregulates Cl-dependent HCO3 secretion, while the SCFA isobutyrate inhibits both cAMP-induced and Cl-dependent HCO3 secretion.



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Fig. 9. Cellular model of HCO3 secretion in colonocytes of rat distal colon. Three distinct modes of HCO3 secretion were identified in rat distal colon: 1) Cl-dependent HCO3 secretion is mediated by apical membrane Cl/HCO3 exchange; the specific apical membrane protein responsible for Cl/HCO3 exchange is uncertain, is indicated as AE? (anion exchange), and could represent either AE1 or DRA (downregulated in adenoma. 2) SCFA-dependent HCO3 secretion is mediated by apical membrane SCFA-HCO3 exchange whose molecular identity has not been established. 3) cAMP-stimulated HCO3 secretion is mediated by apical membrane CFTR. cAMP also inhibited Cl-dependent HCO3 secretion, while SCFA downregulated both Cl-dependent HCO3 secretion and cAMP-induced HCO3 secretion.

 

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These studies were supported in part by United States Public Health Service Grant DK-60069 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Wellcome Trust/Burroughs Wellcome Fund.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Binder, Dept. of Internal Medicine, Yale Univ., PO Box 208019, New Haven, CT 06520 (E-mail: henry.binder{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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