Departments of 1Internal Medicine, 2Cellular and Molecular Physiology, and 3Surgery, Yale University, New Haven, Connecticut
Submitted 15 September 2004 ; accepted in final form 14 January 2005
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ABSTRACT |
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Cl/HCO3 exchange; short-chain fatty acid/HCO3 exchange; anion channel; pH stat; colonic mucosa
In vivo perfusion of the colon with SCFA has been associated with HCO3 secretion (1, 2, 34, 41). Although relatively little is known about the SCFA-dependent HCO3 secretion, recent studies indicate that SCFA induce HCO3 secretion via a Cl-independent, DIDS-insensitive process that probably involves an apical membrane SCFA/HCO3 exchange (42). Because SCFA could stimulate HCO3 secretion as a result of their membrane transport and/or metabolism, these initial studies were performed with a poorly metabolized SCFA, isobutyrate (42). Additional studies of HCO3 secretion with butyrate would be required to establish the relative contribution of metabolism and transport to overall SCFA-dependent HCO3 secretion.
At least three different modes of HCO3 secretion in the rat distal colon have been identified as 1) Cl-dependent, 2) cAMP-induced, and 3) SCFA-dependent, which have been characterized by their distribution to surface and crypt cells, dependence on lumen Cl, and response to 100 µM DIDS, an inhibitor of Cl/anion exchange, and to 100 µM 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a nonspecific inhibitor of anion channels (42). Cl-dependent HCO3 secretion is a DIDS-sensitive and NPPB-insensitive process located in surface epithelial but not in crypt epithelial cells. In contrast, cAMP-induced HCO3 secretion is both Cl-independent and DIDS-insensitive but is NPPB-sensitive, located in crypt and possibly in surface epithelial cells (13). The exact site for SCFA-dependent HCO3 secretion is not known, but because SCFA absorption is presumably a transport process located in surface cells, SCFA-dependent HCO3 secretion is likely present in surface cells. Furthermore, SCFA-dependent HCO3 secretion has recently been established as Cl insensitive and both DIDS and NPPB insensitive (42). SCFA have also been shown to inhibit both cAMP-induced and Cl-dependent HCO3 secretion, but little is known about the mechanism of this downregulation.
These present studies were designed to explore in detail the mechanism of butyrate-dependent HCO3 secretion with emphasis on the role of both SCFA transport and metabolism in the genesis of colonic HCO3 secretion; the cellular site(s) of SCFA-dependent HCO3 secretion; and the interrelationship between SCFA and both Cl-dependent and cAMP-induced HCO3 secretion.
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MATERIALS AND METHODS |
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pH Stat Technique
HCO3 secretion was determined by pH stat methods recently described in detail (42). In brief, colonic mucosa stripped of serosal and muscular layers were mounted in Lucite chambers under voltage clamp conditions. The standard serosal solution contained (in mM) 140 Na, 5.2 K, 119.8 Cl, 25 HCO3, 2.4 HPO4, 0.4 H2PO4, 1.2 Ca, 1.2 Mg. Colonic tissues were always exposed to a buffered solution on the serosal side, whereas the lumen side was always a HCO3-free, low-buffered solution (0.1 mM HEPES-Tris buffer, pH 7.4). Composition of the several solutions used in these experiments is presented in Table 1 and depended on the specific experiment being performed. HCO3-containing solutions were gassed with 95% O2-5% CO2, and the HCO3-free solutions were gassed with 100% O2. In isobutyrate kinetic studies (025 mM) isosmolarity was maintained by varying concentration of isethionate. All experimental solutions were calibrated by using a known quantity of 0.025 N H2SO4 added against known concentration of HCO3 to obtain a linear curve. The determined quantity of HCO3 was always within the linear range of this curve for all lumen solutions. The pH stat technique involved a constant maintenance of pH by addition of 0.025 N H2SO4. HCO3 secretion was defined as the amount of acid required to maintain a constant (or stat) pH over a period of time and is expressed as µeq·h1·cm2. HCO3 secretion was constant for at least 80 min.
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Crypt Microperfusion
Individual crypts were obtained from the distal colon by hand dissection. Net fluid movement was determined by using microperfusion technology with 3H-labeled inulin, as previously described (13). Results are expressed as nl·mm1·min1. HCO3 secretion was determined from net fluid movement and HCO3 concentration ([HCO3]) in both perfusate and effluent. [HCO3] was measured by thermogenic determination using a picapnotherm previously standardized in this laboratory (13). Net HCO3 movement is expressed as picamoles per millimeter per minute. Each data point represents the average of three 5-min collections of effluent. At least five crypts were studied in each experiment. At the end of the experiment, the viability of the crypt was assessed with trypan blue, and experiments in which cells failed to exclude dye were discarded. Positive values represent net absorption, and negative ones, net secretion. Solutions used with microperfusion were equilibrated with 5% CO2 and adjusted to an osmolality of 300310 osmol/kg. In the nominally CO2/HCO3-free solutions, 32 mM HEPES replaced 22 mM NaHCO3 and the solution was air equilibrated. The pH of all solution was 7.4 at 37°C.
Vesicle Preparation and Uptake Studies
Apical membrane vesicles (AMV) were prepared from both surface and crypt cells of normal rat distal colon, as previously described (28). Surface cell AMV was prepared by the method of Stieger et al. (39). Crypt cell AMV was isolated by the method of Lomax et al. (21). Initial rate of 14C-butyrate (Amersham, Arlington Heights, IL), 14C-labeled isobutyrate (American Radiolabeled Chemicals, St. Louis, MO), and 36Cl (New England Nuclear, Boston, MA) uptake was performed by rapid filtration techniques, as previously presented (27). In brief, in studies to delineate butyrate/HCO3 exchange, AMV were preloaded with either 50 mM HEPES-Tris (pH 7.5) containing 150 mM KHCO3 or 50 mM HEPES-Tris (pH 7.5) containing 150 mM K-gluconate. Uptake was measured by incubating the AMV for 3 s in medium containing 50 mM MES-Tris (pH 6.0), 25 µM 14C-labeled butyrate (or [14C]-labeled isobutyrate) and 150 mM K gluconate.
Butyrate/HCO3 exchange. Butyrate/HCO3 exchange measured as outward [HCO3] gradient-driven butyrate uptake was calculated by subtracting the uptake in AMV preloaded with gluconate from that in AMV preloaded with HCO3.
Butyrate/butyrate exchange. Butyrate/butyrate exchange was determined in AMV preloaded with 50 mM HEPES-Tris (pH 7.5) containing 150 mM Na-butyrate. Uptake was measured by incubating the AMV for 2 s in medium containing 50 mM MES-Tris (pH 6.0), 25 µM 14C-butyrate, and 150 mM of either Na-gluconate or Na-butyrate. Outward butyrate concentration ([butyrate]) and gradient-driven butyrate uptake (i.e., butyrate/butyrate exchange) was calculated by subtracting the uptake in incubation medium with butyrate from that in incubation with gluconate.
Cl/butyrate exchange. Cl/butyrate exchange was determined in AMV preloaded with 50 mM MES-Tris (pH 6.5) containing (in mM) 50 Na-gluconate, 100 K-gluconate, and 10 NMG-gluconate. Uptake was measured by incubating the AMV for 9 s in medium containing (in mM) 50 MES-Tris (pH 5.5), 50 Na-gluconate, 100 K-gluconate, and 10 NMG-36Cl. Uptake was also performed in the presence of 1 mM DIDS. Outward [butyrate] gradient-driven Cl uptake (i.e., Cl/butyrate exchange) was calculated by subtracting the uptake in medium with DIDS from that in the absence of DIDS.
Cl/HCO3 exchange. Cl/HCO3 exchange was determined in AMV preloaded with 50 mM HEPES-Tris (pH 7.5) containing (in mM) 150 KHCO3, 20 Na-gluconate, and 10 NMG-gluconate. Uptake was measured for 12 s by incubating the AMV in medium containing (in mM) 50 HEPES-Tris (pH 7.5), 20 Na-gluconate, 10 NMG-36Cl, and 150 of either HCO3 or K-gluconate. Uptake in the presence of extravesicular 20 mM Na-butyrate was also measured by replacing Na-gluconate. Outward [HCO3] gradient-driven Cl uptake (i.e., Cl/HCO3 exchange) was calculated by subtracting the uptake in medium with HCO3 from that in medium with gluconate.
Cl/Cl exchange. Cl/Cl exchange was determined in AMV preloaded with 50 mM HEPES-Tris (pH 7.5) containing (in mM) 150 KCl, 20 Na-gluconate, and 10 NMG-gluconate. Uptake was measured for 12 s by incubating the AMV in medium containing (in mM) 50 HEPES-Tris (pH 7.5), 20 Na-gluconate, 10 NMG-36Cl, and 150 of either KCl or K-gluconate. Uptake in the presence of extravesicular 20 mM Na-butyrate was also measured by replacing Na-gluconate. Outward Cl concentration ([Cl]) gradient-driven Cl uptake (i.e., Cl/Cl exchange) was calculated by subtracting the uptake in medium with K-gluconate from that in medium with KCl.
Cl/OH exchange. Cl/OH exchange was determined in AMV preloaded with 50 mM HEPES-Tris (pH 7.5) containing (in mM) 150 K-gluconate, 20 Na-gluconate, and 10 NMG-gluconate. Uptake was measured for 12 s by incubating the AMV in medium containing (in mM) 150 K-gluconate, 20 Na-gluconate, 10 NMG-36Cl and 50 of either HEPES-Tris (pH 7.5) or MES-Tris (pH 5.5). Uptake in the presence of extravesicular 20 mM Na-butyrate was also measured by replacing Na-gluconate. Outward OH concentration ([OH]) gradient-driven Cl uptake (i.e., Cl/OH exchange) was calculated by subtracting the uptake in medium with HEPES-Tris (pH 7.5) from that in medium with MES-Tris (pH 5.5).
Cl channel activity. Cl channel activity was determined in AMV isolated from crypt cells preloaded with 50 mM HEPES-Tris (pH 7.5) containing 160 mM NMG-gluconate and 20 mM Na-gluconate. Intravesicular positive membrane potential-driven uptake was measured for 9 s by incubating the AMV in medium containing (in mM) 50 HEPES-Tris (pH 7.5), 150 of K-gluconate, 10 NMG-36Cl, and 20 of either Na-gluconate or Na-butyrate, plus 25 µM valinomycin,. Uptake was also performed in the presence of 100 µM NPPB. Electrogenic anion channel activity (i.e., Cl channel activity) was calculated by subtracting the uptake in medium with NPPB from that in the absence of NPPB.
Chemicals
5-acetamido-1,3,4-thiadiazole-2-sulfonamide (acetazolamide), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), 2-bromo octanoic acid, DIDS, butyrate, DMSO grade I, HEPES, isethionic acid, 2-methylpropionic acid (isobutyric acid), N-methyl-D-glucamine, NPPB, potassium gluconate, and sodium gluconate were obtained from Sigma (St. Louis, MO).
Statistics
Results are presented as means ± SE. Statistical analyses were performed by using paired and unpaired t-test and Bonferroni's one-way ANOVA post hoc test. P < 0.05 was considered significant.
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RESULTS |
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Effect of butyrate on HCO3 secretion. Our prior experiments of SCFA-dependent HCO3 secretion were performed with isobutyrate, a relatively poorly metabolized SCFA (42). The present studies sought to characterize the effect of lumen butyrate on HCO3 secretion because butyrate is normally present in large amounts in stool and is both transported and metabolized by the large intestine. Table 2 presents the results of butyrate-dependent HCO3 secretion indicating that the characteristics of butyrate-dependent HCO3 secretion are identical to those initially reported for lumen isobutyrate (42). That is, butyrate-dependent HCO3 secretion was not altered by the removal of lumen Cl or by the addition of either lumen DIDS (100 µM) or lumen NPPB (100 µM). Our prior studies with lumen isobutyrate demonstrated that both Cl-dependent HCO3 secretion and cAMP-induced HCO3 secretion were downregulated by 25 mM isobutyrate (42). The 25 mM lumen butyrate also inhibited both Cl-dependent and cAMP-induced HCO3 secretion (data not shown). Neither 25 mM butyrate nor 25 mM isobutyrate added to the serosal solution resulted in any change in the low rate of basal HCO3 secretion (data not shown). These observations suggest that SCFA-dependent HCO3 secretion is closely linked to SCFA transport across the luminal membrane.
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Effect of lumen isobutyrate on Cl-dependent HCO3 secretion.
Prior studies demonstrated that 25 mM isobutyrate completely inhibited Cl-dependent HCO3 secretion (42). A series of studies were designed to determine the concentration dependence of this inhibition. Cl-dependent HCO3 secretion in the absence of isobutyrate is completely DIDS-sensitive. Addition of increasing concentrations of isobutyrate up to 25 mM in the presence of 119.8 mM Cl did not alter the rate of total HCO3 secretion (Fig. 1). In contrast, a striking qualitative difference was seen when the total HCO3 secretion was analyzed as the DIDS-sensitive and DIDS-insensitive components. Although 1 mM isobutyrate did not affect DIDS-sensitive HCO3 secretion in the presence of lumen Cl, increasing the concentration of isobutyrate (while maintaining Cl concentration constant) revealed an increasing rate of the DIDS-insensitive component of HCO3 secretion. The DIDS-sensitive component of HCO3 secretion progressively decreased as the isobutyrate concentration was increased. Thus at 3 mM isobutyrate, HCO3 secretion was 20% DIDS-insensitive, whereas at 4 mM isobutyrate, 75% of HCO3 secretion was DIDS-insensitive. HCO3 secretion was completely DIDS-insensitive at 6 mM isobutyrate. These results indicate that 6 mM isobutyrate had both inhibited Cl-dependent DIDS-sensitive HCO3 secretion and induced isobutyrate-dependent DIDS-insensitive HCO3 secretion.
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Effect of lumen isobutyrate on cAMP-induced HCO3 secretion. Because 25 mM isobutyrate also inhibited cAMP-induced HCO3 secretion, parallel experiments were performed to establish the relative isobutyrate concentrations that inhibited cAMP-induced HCO3 secretion. Addition of increasing concentrations of isobutyrate in the presence of cAMP did not significantly affect the total HCO3 secretion but altered the relative NPPB-sensitive and NPPB-insensitive component of HCO3 secretion (Fig. 2). Inhibiting HCO3 secretion with NPPB revealed that NPPB-sensitive component decreased, whereas the NPPB-insensitive component increased as lumen isobutyrate concentration was increased. In the absence of isobutyrate, cAMP-induced HCO3 secretion is 100% NPPB-sensitive. The addition of 1 mM isobutyrate reduced NPPB-sensitive cAMP-induced HCO3 secretion by 25% (Fig. 2), whereas 2 mM isobutyrate decreased NPPB-sensitive cAMP-induced HCO3 secretion by 40%. Increasing isobutyrate to 12.5 mM produced only a modest further increase in inhibition of NPPB-sensitive cAMP-induced HCO3 secretion. In contrast, 15 mM isobutyrate reduced NPPB-sensitive cAMP-induced HCO3 secretion by 80%, whereas 20 mM isobutyrate completely inhibited NPPB-sensitive cAMP-induced HCO3 secretion.
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Microperfusion Studies
These pH stat studies demonstrated the presence of butyrate-dependent HCO3 secretion. pH stat studies will presumably identify HCO3 secretion that originated in surface and/or crypt cells but cannot distinguish between these two cell populations. Therefore, to establish the specific site of butyrate-dependent HCO3 secretion, microperfusion experiments were performed with isolated colonic crypts. As previously reported (13), net HCO3 absorption was present in the control period in the presence or absence of lumen Cl. Addition of either 10 or 20 mM butyrate to the lumen perfusion solution did not change the rate of net HCO3 absorption (Fig. 4A). At no time was net HCO3 secretion observed. The effect of butyrate on net fluid movement was also examined. As previously demonstrated (37), net fluid absorption was present in the basal period and remained unaltered after perfusion with lumen 10 or 20 mM butyrate (data not shown). These observations establish that neither butyrate-dependent HCO3 secretion nor butyrate stimulation of fluid absorption are present in crypt cells, and, as a result, these observations indicate that the butyrate-dependent HCO3 secretion observed in the pH stat experiments must arise from surface and not from crypt cells.
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Apical Membrane Vesicle Studies
Prior studies (23) established the presence of butyrate/HCO3 exchange in AMV prepared from the distal colon of rats. To determine the cell-specific localization of butyrate/HCO3 exchange, the effect of an outward-directed [HCO3] gradient on 14C-butyrate uptake by AMV prepared from isolated surface epithelial and crypt epithelial cells was examined. In these studies, the initial rate of outward [HCO3] gradient-driven 14C-butyrate uptake (Fig. 5, left) and outward [butyrate] gradient-driven 14C-butyrate uptake (Fig. 5, right) was measured as evidence for butyrate/HCO3 exchange in AMV isolated from surface and crypt cells. Both [HCO3] gradient- and [butyrate] gradient-driven 14C-butyrate uptake was present in AMV isolated from surface cells (Fig. 5). In contrast, neither [HCO3] gradient- nor [butyrate] gradient-driven 14C-butyrate uptake was present in AMV from crypt cells. Additional studies were also performed to examine the integrity of the butyrate transport in AMV from crypt cells. In these studies outward [butyrate] gradient-driven 36Cl uptake was used as evidence for Cl/butyrate exchange. As shown in Fig. 6, [butyrate] gradient-driven 36Cl uptake was present in AMV from both surface and crypt cells, indicating that changes in membrane integrity as a result of membrane preparation were not responsible for the failure to observe either butyrate-HCO3 or butyrate-butyrate transport function in these AMV prepared from crypt cells. These observations indicate that butyrate/HCO3 exchange is selectively present in apical membranes of surface epithelial cells, but is not expressed in apical membranes of crypt epithelial cells consistent with the absence of butyrate-dependent HCO3 secretion in the microperfusion studies (Fig. 4A).
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Studies were also designed to establish whether butyrate directly or indirectly inhibits Cl-dependent (i.e., Cl/HCO3 exchange) and cAMP-induced (i.e., anion channel) HCO3 secretion. In these studies, the effect of 20 mM butyrate was examined on Cl/anion exchanges (i.e., Cl/HCO3, Cl/Cl, and Cl/OH exchanges) and electrogenic Cl channels (Figs. 7 and 8). As shown in Fig. 7A, extravesicular presence of 20 mM butyrate almost completely (98%) inhibited Cl/HCO3 exchange. Because these AMV also contain butyrate/HCO3 exchange, it is possible that extravesicular butyrate could have inhibited Cl/HCO3 exchange by dissipating the HCO3 gradients (Fig. 5 and Ref. 26). Therefore, the effect of butyrate on Cl/Cl exchange was also examined. As shown in Fig. 7B, Cl/Cl exchange was only partially inhibited (68%) by extravesicular butyrate. Because distinct and separate Cl/HCO3 and Cl/OH exchanges that could function as Cl/Cl exchange are present in these AMV from distal colon, the effect of butyrate on Cl/OH exchange was also examined. As shown in Fig. 7C, Cl/OH exchange was only partially inhibited (40%) by extravesicular butyrate. In contrast, formate did not inhibit either Cl/HCO3 exchange, Cl/Cl exchange, or Cl/OH exchange (data not shown). In additional studies, the effect of extravesicular butyrate on apical membrane electrogenic anion channels was examined by determining potential-dependent 36Cl movement in AMV prepared from crypt cells. As shown in Fig. 8, an intravesicular positive potential generated by inward K gradient and valinomycin stimulated NPPB (a nonspecific anion channel blocker)-sensitive 36Cl uptake in AMV isolated from crypt cells. The presence of extravesicular butyrate almost completely inhibited (96%) Cl channel activity. These observations suggest that butyrate directly inhibits both apical membrane Cl/HCO3 exchange and electrogenic anion channels.
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DISCUSSION |
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Additional effects of SCFA on colonic ion transport have been identified. First, butyrate inhibits cAMP- and cGMP-stimulated Cl secretion, but not Cai-mediated Cl secretion (6, 10). Second, although the mechanism of both HCO3-dependent and butyrate-dependent Na absorption require an NHE and cAMP inhibits both NHE and HCO3-dependent Na absorption, butyrate-dependent Na absorption is not altered by cAMP (3, 4). Third, in the presence of increased mucosal cAMP, butyrate-dependent Na absorption is enhanced as a consequence of an increase in Vmax without any change in Km (19). Subsequent studies established that HCO3-dependent Na absorption requires NHE-3 isoform and not NHE-2 isoform. In contrast, either NHE-2 or NHE-3 isoform is linked to butyrate-dependent Na absorption, and cAMP inhibits NHE-3 isoform but activates NHE-2 isoform. (18). Fourth, SCFA not only stimulate HCO3 secretion but also inhibit both Cl-dependent and cAMP-induced HCO3 secretion (Table 2, Figs. 1, 2 and Ref. 42), whereas the experiments presented in Figs. 7 and 8 provide evidence that SCFA directly inhibits both Cl/HCO3 exchange and electrogenic anion channel activity.
Bicarbonate secretion is an essential intestinal function and is often associated with fluid loss in diarrhea. Despite its critical role, HCO3 secretion has not been studied as extensively as either Cl secretion or Na absorption in either small or large intestine (11, 35, 36). During the past several years duodenal HCO3 secretion has been explored by several investigators primarily in relationship with the mechanism of neutralization of gastric acid secretion (8, 16). These studies, as well as those previously performed in the ileum and colon (12, 24, 35, 36, 38, 40), suggested that the mechanism of HCO3 secretion is primarily Cl-dependent and closely linked to an apical membrane Cl/HCO3 exchange (12, 24, 36). Because cAMP also induces HCO3 secretion, there has been evidence that cAMP-induced HCO3 secretion may occur via an anion channel (possibly CFTR) or via Cl/HCO3 exchange coupled to an anion channel (8, 16, 17, 35).
During the past few years this laboratory has studied HCO3 secretion by two different methodological approaches: 1) microperfusion of isolated crypts with direct determination of HCO3 (13, 37) and 2) colonic mucosa stripped of muscular layers in Lucite chambers with pH stat quantitation of HCO3 (42). The latter studies identify HCO3 secretion that may have originated in surface and/or crypt cells. The crypt microperfusion studies demonstrated that cAMP induced HCO3 secretion in crypt epithelial cells (13); in contrast, neither Cl-dependent nor butyrate-dependent HCO3 secretion was identified during crypt microperfusion (Fig. 4A and Ref. 13). This spatial distribution of Cl-dependent and butyrate-dependent HCO3 correlates closely with the distribution of both Cl/HCO3 exchange and butyrate/HCO3 exchange in AMV of surface epithelial cells but not in those of crypt epithelial cells (Fig. 5 and Ref. 26). In contrast, Cl/butyrate exchange that is most likely not associated with either Cl-dependent or SCFA-dependent HCO3 secretion is present in apical membranes of both surface and crypt cells (Fig. 6). These present studies also provide evidence that Cl-dependent HCO3 secretion is a result of apical membrane Cl/HCO3 exchange and not Cl/OH exchange. First, Cl/OH exchange is present in apical membranes of both surface and crypt cells (26), and 25 mM butyrate almost completely inhibits both Cl/HCO3 exchange in AMV (Fig. 7A) and Cl-dependent HCO3 secretion (Fig. 1), but only results in a 40% downregulation of Cl/OH exchange (Fig. 7C).
Identification of both Cl-dependent and butyrate-dependent HCO3 secretion in the pH stat studies (Ref. 42 and Table 2) but not in the crypt microperfusion experiments (13 and Fig. 4A) provide compelling evidence that these two HCO3 secretory mechanisms are present in surface, but not in crypt epithelial cells. In contrast, both the crypt microperfusion studies and pH stat experiments identified cAMP-induced HCO3 secretion, but it is not definitely known whether cAMP-induced HCO3 secretion is present in both surface and crypt epithelial cells or only in crypt epithelial cells. Analyses of the present results provide strong evidence that the HCO3 secretion identified in pH stat studies primarily represents a surface cell and not a crypt cell process. This conclusion is based on combined demonstration that butyrate failed to downregulate cAMP-induced HCO3 secretion in the microperfusion studies (Fig. 4B), whereas in the pH stat studies butyrate inhibited cAMP-induced HCO3 secretion (Fig. 2). Thus the components of HCO3 secretion observed in the pH stat experiments (i.e., Cl-dependent, SCFA-dependent and cAMP-induced) represent HCO3 transport processes located in surface epithelial and not in crypt epithelial cells. The present results also suggest that cAMP-induced HCO3 secretion is present in both surface and crypt cells but with varying degrees of sensitivity to butyrate because butyrate inhibits cAMP-induced HCO3 secretion in the pH stat studies (Fig. 2) but not in the microperfusion experiments (Fig. 4B). These observations permit the speculation that regulation of cAMP-induced HCO3 secretion by SCFA differs in surface and crypt cells.
Comparison of butyrate-dependent and isobutyrate-dependent HCO3 secretion experiments suggests that butyrate induces HCO3 secretion as a result of both SCFA transport and SCFA metabolism. The conclusion that the primary mechanism of SCFA-dependent HCO3 secretion is a result of SCFA transport via the apical membrane SCFA/HCO3 exchange is based on several observations. First, HCO3 secretion was stimulated by lumen isobutyrate (Table 3), but not by serosal 25 mM isobutyrate (0.5 ± 0.08 µeq·h1·cm2). Second, isobutyrate is a poorly metabolized SCFA, and isobutyrate-dependent HCO3 secretion is completely dependent on bath HCO3 and is only modestly (25%) inhibited by acetazolamide (Table 3). Third, high concentrations (i.e., 25 mM) of butyrate that are metabolized by colonic epithelial cells induced HCO3 secretion that has both a serosal HCO3-independent component and was substantially reduced both by carbonic anhydrase inhibition (57%) (Table 3) and by 2-bromo-octonoate, an inhibitor of butyrate metabolism. Indeed, the acetazolamide-insensitive component of isobutyrate-dependent HCO3 secretion is almost identical to the acetazolamide-insensitive component of butyrate-dependent HCO3 secretion (4.3 ± 0.05 vs. 3.6 ± 0.04 µeq·h1·cm2) (Table 3). Fourth, at 1 mM butyrate and isobutyrate the effects of bath HCO3 and acetazolamide are essentially identical, indicating that at low SCFA concentrations the stimulation of HCO3 secretion is primarily linked to SCFA transport. These observations support the conclusion that butyrate-dependent HCO3 secretion is a consequence largely but not exclusively of butyrate transport represented by the component of HCO3 secretion that is serosal HCO3-dependent but insensitive to acetazolamide. A smaller fraction of HCO3 secretion is linked to butyrate metabolism, which is the component of HCO3 secretion that is serosal HCO3-insensitive but sensitive to inhibition of carbonic anhydrase inhibition and butyrate metabolism. It is not unlikely that a small fraction of butyrate-dependent HCO3 secretion is linked to either butyrate transport or metabolism. The relative importance of butyrate transport on HCO3 secretion is substantially greater at low butyrate concentrations than at higher ones. Finally, because the initial rate of butyrate/HCO3 exchange was 35% greater than that of isobutyrate/HCO3 exchange, the higher rate of butyrate-stimulated than isobutyrate-stimulated HCO3 secretion was a result of both butyrate transport and metabolism.
Mechanism(s) by which SCFA inhibit C1-dependent and cAMP-induced HCO3 secretion were suggested by our present experiments. One possibility is that the intracellular presence of SCFA results in a decrease in intracellular pH and that both Cl-dependent and cAMP-stimulated HCO3 secretory processes are pH sensitive. An alternate possibility is that the affinity of these two inhibited HCO3 secretory processes for HCO3 is less than that of SCFA/HCO3 exchange. A third possibility is that SCFA directly inhibits the apical membrane transport process responsible for HCO3 secretion. The experiments presented in Figs. 7 and 8 provide support for this suggestion by demonstrating that butyrate almost completely inhibited both apical membrane Cl/HCO3 exchange and apical membrane anion channel activity. Furthermore, the effect of SCFA on these two HCO3 transporters is not nonspecific, because butyrate did not affect NHE activity and formate did not alter Cl/anion exchange in AMV (V. M. Rashendran and H. J. Binder, unpublished observations). The failure of SCFA to inhibit cAMP-induced HCO3 secretion in colonic crypts (Fig. 4B) is not readily apparent, but may be a result of different mechanisms of SCFA movement across the basolateral membrane or of different cellular SCFA metabolism in surface and crypt cells.
Presence of both SCFA-dependent HCO3 secretion and apical membrane SCFA/HCO3 exchange in surface but not crypt cells, and the demonstration that both SCFA-dependent HCO3 secretion and SCFA/HCO3 exchange are DIDS-insensitive suggest that SCFA-dependent HCO3 secretion is related to an apical membrane SCFA/HCO3 exchange. The molecular identity of this apical membrane transporter is not known. Recent studies (9, 14, 3032) with pig and human colon and human colonic cell lines (CaCo2 and AA/C1) indicate that MCT1 is an apical membrane SCFA transport mechanism. Whereas MCT1-mediated SCFA transport has been proposed both as SCFA-H+ cotransport and as SCFA/HCO3 exchange (14, 30, 32), inhibition of SCFA-dependent HCO3 secretion by removal of serosal HCO3 (Table 3) is not consistent with SCFA-H+ cotransport. Moreover, MCT1-mediated SCFA transport is inhibited by 4-hydroxy cinnamate (4CHC), an inhibitor of monocarboxylate transporters (14, 32). Because HCO3 gradient-driven 14C-butyrate uptake by AMV from rat distal colon is not affected by 4CHC (V. M. Rashendran and H. J. Binder, unpublished observations), it is unlikely that MCT1 mediates SCFA-dependent HCO3 secretion in the rat distal colon.
These studies have explored the effect of SCFA on colonic HCO3 secretion and have made possible several conclusions. First, SCFA-induced HCO3 secretion is the result of both SCFA transport across the apical membrane via a SCFA/HCO3 exchange and intracellular metabolism of SCFA. Second, SCFA-induced HCO3 secretion represents a surface epithelial cell process because butyrate-dependent HCO3 secretion was observed in the pH stat studies but not in the crypt microperfusion experiments and butyrate/HCO3 exchange was identified in apical membranes of surface but not crypt epithelial cells. Third, SCFA downregulates both Cl-dependent and cAMP-induced HCO3 secretion with the inhibition of Cl-dependent HCO3 secretion linked to direct interaction of SCFA with an apical membrane anion exchange (Fig. 7). In contrast, SCFA inhibition of cAMP-induced HCO3 secretion appears dependent primarily on its direct downregulation of the apical membrane anion channel (Fig. 8). We, therefore, conclude that as SCFA are normally present in the large intestine in significant amounts and lumen SCFA inhibit both Cl-dependent and cAMP-induced HCO3 secretion, SCFA-dependent HCO3 secretion is likely the predominant mechanism of HCO3 secretion in the mammalian colon.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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