Role of short-chain fatty acids in colonic HCO3 secretion

Sadasivan Vidyasagar,1 Christian Barmeyer,1 John Geibel,2,3 Henry J. Binder,1,2 and Vazhaikkurichi M. Rajendran1

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Luminal isobutyrate, a relatively poor metabolized short-chain fatty acid (SCFA), induces HCO3 secretion via a Cl-independent, DIDS-insensitive, carrier-mediated process as well as inhibiting both Cl-dependent and cAMP-induced HCO3 secretion. The mechanism(s) responsible for these processes have not been well characterized. HCO3 secretion was measured in isolated colonic mucosa mounted in Lucite chambers using pH stat technique and during microperfusion of isolated colonic crypts. 14C-labeled butyrate, 14C-labeled isobutyrate, and 36Cl uptake were also determined by apical membrane vesicles (AMV) isolated from surface and/or crypt cells. Butyrate stimulation of Cl-independent, DIDS-insensitive 5-nitro-3-(3-phenylpropyl-amino)benzoic acid-insensitive HCO3 secretion is greater than that by isobutyrate, suggesting that both SCFA transport and metabolism are critical for HCO3 secretion. Both lumen and serosal 25 mM butyrate inhibit cAMP-induced HCO3 secretion to a comparable degree (98 vs. 90%). In contrast, Cl-dependent HCO3 secretion is downregulated by lumen 25 mM butyrate considerably more than by serosal butyrate (98 vs. 37%). Butyrate did not induce HCO3 secretion in isolated microperfused crypts, whereas an outward-directed HCO3 gradient-driven induced 14C-butyrate uptake by surface but not crypt cell AMV. Both 36Cl/HCO3 exchange and potential-dependent 36Cl movement in AMV were inhibited by 96–98% by 20 mM butyrate. We conclude that 1) SCFA-dependent HCO3 secretion is the result of SCFA transport across the apical membrane via a SCFA/HCO3 exchange more than intracellular SCFA metabolism; 2) SCFA-dependent HCO3 secretion is most likely a result of an apical membrane SCFA/HCO3 exchange in surface epithelial cells; 3) SCFA downregulates Cl-dependent and cAMP-induced HCO3 secretion secondary to SCFA inhibition of apical membrane Cl/HCO3 exchange and anion channel activity, respectively.

Cl/HCO3 exchange; short-chain fatty acid/HCO3 exchange; anion channel; pH stat; colonic mucosa


SHORT-CHAIN FATTY ACIDS (SCFA) are the primary anion in stool, are not normal dietary constituents, but are synthesized from nonabsorbed carbohydrates by colonic bacteria (5). Of the numerous SCFA, acetate, propionate, and butyrate have the highest fecal concentrations. Butyrate is often considered the most important because it is the principal colonocyte nutrient (33). Critical roles for SCFA have been identified in both health and disease, and SCFA have multiple effects on ion transport in colonocytes (6, 10, 18, 20).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male Sprague-Dawley rats weighing ~200–250 g were used in all experiments. Rats were killed by exsanguination. The experimental protocols described in this communication were approved by the Yale University Institutional Animal Care and Use Committee.

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 (0–25 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·h–1·cm2. HCO3 secretion was constant for at least 80 min.


View this table:
[in this window]
[in a new window]
 
Table 1. Composition of experimental solutions

 
Because HCO3 secretion was defined as the amount of acid required to maintain the pH at 7.4, prior studies evaluated the possibility that parallel proton secretion, i.e., secondary to Na+/H+ exchange (NHE) and H+-K+-ATPase, was present, which had resulted in an underestimation of HCO3 secretion (42). In those studies, removal of lumen Na, addition of lumen 1 mM amiloride, or addition of lumen 1 mM amiloride plus removal of lumen Na did not increase HCO3 secretion. Because these studies suggested the absence of a functional NHE, unidirectional 22Na fluxes were performed to determine net Na absorption (JnetNa). Because these experiments demonstrated an absence of JnetNa, it was concluded that the serosal-to-lumen HCO3 gradient was responsible for the absence of JnetNa. In additional studies, the addition of 1 mM orthovanadate, an inhibitor of ATPases, resulted in a 7% increase in HCO3 secretion. As a result, use of the pH stat method to determine HCO3 secretion does not substantially underestimate actual rate of HCO3 secretion secondary to unrecognized proton secretion (42).

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·mm–1·min–1. 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 300–310 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
pH Stat Studies

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.


View this table:
[in this window]
[in a new window]
 
Table 2. Characterization of butyrate-dependent HCO3 secretion

 
Effect of bath HCO3 and acetazolamide on butyrate-dependent and isobutyrate-dependent HCO3 secretion. Although butyrate-dependent and isobutyrate-dependent HCO3 secretion were qualitatively identical, the rate of 25 mM butyrate-dependent HCO3 secretion was significantly greater (47%) than that of 25 mM isobutyrate-dependent HCO3 secretion (8.2 ± 0.03 vs. 5.7 ± 0.05 µeq·h–1·cm–2) (Table 3). Isobutyrate-dependent HCO3 secretion was completely inhibited by the removal of serosal HCO3, indicating that an extracellular source of HCO3 was required for isobutyrate-dependent HCO3 secretion. This experiment also excluded the possibility that the luminal alkalinization observed in these experiments represented proton absorption via either H+-SCFA cotransport or nonionic diffusion of SCFA. In contrast, the removal of serosal HCO3 resulted in only a 77% inhibition of butyrate-dependent HCO3 secretion; the component of butyrate-dependent HCO3 secretion that was serosal HCO3-independent was 1.9 ± 0.01 µeq·h–1·cm–2, indicating that the source of HCO3 secreted in the presence of lumen butyrate was primarily but not solely derived from serosal HCO3. Because an intracellular source of HCO3 was likely linked to the intracellular production of HCO3 by carbonic anhydrase, experiments were performed with acetazolamide, a carbonic anhydrase inhibitor. Table 3 also demonstrates that butyrate-dependent HCO3 secretion was substantially inhibited by acetazolamide (3.6 ± 0.04 vs. 8.4 ± 0.03 µeq·h–1·cm–2). In contrast, acetazolamide only modestly reduced isobutyrate-dependent HCO3 secretion (4.3 ± 0.5 vs. 5.7 ± 0.05 µeq·h–1·cm–2). In addition, the component of butyrate-dependent HCO3 secretion observed in the absence of serosal HCO3 was completely inhibited by the addition of acetazolamide. The acetazolamide-insensitive component of butyrate-dependent HCO3 secretion was almost identical to that of isobutyrate-dependent HCO3 secretion (3.6 ± 0.04 vs. 4.3 ± 0.05 µeq·h–1·cm–2). These observations with acetazolamide indicate that >57% of butyrate-dependent HCO3 secretion is linked to a carbonic anhydrase-mediated process, whereas only 24% of isobutyrate-dependent HCO3 secretion was acetazolamide-sensitive. These results suggest that butyrate-dependent HCO3 secretion is likely the result of both butyrate transport and metabolism. In contrast, isobutyrate-dependent HCO3 secretion is primarily linked to isobutyrate transport.


View this table:
[in this window]
[in a new window]
 
Table 3. Comparison of 25 mM mucosal isobutyrate-dependent and butyrate-dependent HCO3 secretion: role of serosal HCO3 and acetazolamide

 
To determine whether the effect of serosal HCO3 and acetazolamide on butyrate-dependent HCO3 secretion was dependent on the concentration of butyrate used in these experiments, a parallel series of studies were performed with 1 mM lumen butyrate. The effect of both acetazolamide and removal of serosal HCO3 on HCO3 secretion observed in the presence of 1 mM butyrate (Table 4) qualitatively differed from that induced by 25 mM butyrate (Table 3). Removal of serosal HCO3 completely inhibited 1 mM butyrate-dependent HCO3 secretion (0.2 ± 0.02 µeq·h–1·cm–2). Similarly, the rate of HCO3 secretion observed in the presence of 1 mM butyrate was only minimally inhibited by the addition of acetazolamide (1.7 ± 0.05 vs. 2.0 ± 0.09 µeq·h–1·cm–2). In contract to the experiments with 1 and 25 mM butyrate, the results of parallel experiments with 1 and 25 mM isobutyrate were essentially identical (Tables 3 and 4). These results demonstrate that at higher (25 mM) butyrate concentrations, a relatively larger component of butyrate-dependent HCO3 secretion is dependent on carbonic anhydrase inhibition than at lower (1 mM) butyrate concentrations. Thus the stimulation of HCO3 secretion at 1 mM butyrate concentrations is primarily linked to butyrate transport.


View this table:
[in this window]
[in a new window]
 
Table 4. Comparison of 1 mM isobutyrate-dependent and butyrate-dependent HCO3 secretion and role of serosal HCO3 and acetazolamide

 
Effect of 2-bromo-octonoate on butyrate-induced HCO3 secretion. To distinguish between butyrate metabolism and its apical membrane transport in the stimulation of HCO3 secretion, experiments were designed to study the effect of 2-bromo-octanoate on butyrate-dependent HCO3 secretion present in the absence of serosal HCO3. Inhibition of {beta}-oxidation by 2 bromo-octonoate significantly inhibited HCO3 secretion induced by lumen butyrate in the absence of serosal HCO3 (0.6 ± 0.06 vs. 1.9 ± 0.01 µeq·h–1·cm–2). This observation indicates that butyrate metabolism, in addition to butyrate transport, contributes to HCO3 secretion.

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.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Effect of increasing lumen isobutyrate concentrations on Cl-dependent HCO3 secretion. The rate of total HCO3 secretion was measured in the presence of varying isobutyrate concentrations (0 to 25 mM) at a constant lumen Cl (119.8 mM) concentration ({blacktriangleup}). The rate of HCO3 secretion measured in the presence of 100 µM DIDS represents DIDS-insensitive HCO3 secretion ({bullet}). The rate of DIDS-sensitive HCO3 secretion ({circ}) was derived by subtracting DIDS-insensitive HCO3 secretion from that of total HCO3 secretion. Each point represents the mean from four different tissues.

 
A second set of experiments was performed in the presence of both 1 mM Cl and 3 mM isobutyrate, the concentrations of their respective half-maximal concentrations (Kms) for their stimulation of HCO3 secretion (42). We found that 1 mM Cl alone and 3 mM isobutyrate alone resulted in HCO3 secretion that was approximately equal (2.8 ± 0.07 vs. 3.0 ± 0.04 µeq·h–1·cm–2). In the presence of both 1 mM Cl and 3 mM isobutyrate, HCO3 secretion was identical (2.9 ± 0.09 µeq·h–1·cm–2) to that observed when each was present alone. Addition of DIDS inhibited HCO3 secretion to 2.4 ± 0.09 µeq·h–1·cm–2, indicating that in the presence of both lumen Cl and isobutyrate, at their respective Kms, HCO3 secretion was predominantly DIDS-insensitive (83% of total HCO3 secretion) and thus represented SCFA-dependent and not Cl-dependent HCO3 secretion.

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.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Effect of increasing lumen isobutyrate concentrations on cAMP-stimulated HCO3 secretion. The rate of total HCO3 secretion was measured in the presence of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and varying isobutyrate (0 to 25 mM) concentrations ({blacktriangleup}). The rate of HCO3 secretion measured in the presence of 100 µM 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB) represents NPPB-insensitive HCO3 secretion ({bullet}). The rate of NPPB-sensitive HCO3 secretion ({circ}) was derived by subtracting NPPB-insensitive HCO3 secretion from that of total HCO3 secretion. Each point represents mean from 4 different tissues.

 
Effect of serosal butyrate on Cl-dependent and cAMP-induced HCO3 secretion. To evaluate whether the inhibition of Cl-dependent and cAMP-induced HCO3 secretion by butyrate was a function of apical membrane butyrate transport or its intracellular presence and/or metabolism, experiments were performed in which 25 mM butyrate were added to the serosal solution. Fig. 3 demonstrates that the addition of serosal butyrate resulted in a partial (or 35%) inhibition of Cl-dependent HCO3 secretion (3.6 ± 0.1 µeq·h–1·cm–2) and a 90% inhibition of cAMP-induced HCO3 secretion (0.5 ± 0.04 µeq·h–1·cm–2).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Effect of serosal butyrate on Cl-dependent and cAMP-stimulated HCO3 secretion. Both Cl-dependent HCO3 secretion and cAMP-induced HCO3 secretion were determined in the presence and absence of serosal 25 mM butyrate. These results were based on experiments in 4 tissues in each group. *P < 0.001 compared with control group.

 
To further establish whether inhibition of cAMP-stimulated HCO3 secretion was consequent to intracellular butyrate or due to its metabolic products, experiments were performed by using both the relatively poorly metabolized SCFA isoform, isobutyrate, and 2-bromo-octonoate, an inhibitor of butyrate oxidation to {alpha}-hydroxy-butyrate. Serosal isobutyrate also inhibited cAMP-stimulated HCO3 secretion to a similar extent as that of serosal butyrate. Similarly, 300 µM 2-bromo-octonoate added to both lumen and serosal solution did not alter the inhibition of cAMP-induced HCO3 secretion produced by bath 25 mM bath butyrate (0.6 ± 0.06 vs. 0.5 ± 0.04 µeq·h–1·cm–2), indicating that the SCFA inhibition of cAMP-induced HCO3 secretion is not secondary to a process linked to SCFA metabolism. In contrast, because Cl-dependent HCO3 secretion was only partially inhibited by 25 mM bath butyrate (compared to its complete inhibition by 25 mM lumen butyrate), butyrate inhibition of Cl-dependent HCO3 secretion is likely a result of the interaction of butyrate with apical membrane Cl/HCO3 exchange at a luminal site.

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.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. Effect of 20 mM butyrate on net HCO3 movement in isolated microperfused colonic crypts. A: perfusion of crypt with butyrate-free solution resulted in net HCO3 absorption (14). Addition of 20 mM butyrate to the lumen solution did not alter the rate of net HCO3 movement. B: net HCO3 secretion was induced by 8-Br-cAMP added to the serosal solution. Subsequent perfusion with either lumen or serosal 20 mM butyrate did not alter HCO3 secretion. HCO3 secretion was expressed as pmol·mm–1·min–1. These results were based on experiments in 5 crypts.

 
Prior pH stat studies (42) and those presented in Fig. 2 demonstrated that lumen isobutyrate and lumen butyrate both stimulated HCO3 secretion and inhibited cAMP-induced HCO3 secretion. Because butyrate-dependent HCO3 secretion was not present in the crypt, experiments were designed to determine whether butyrate would inhibit cAMP-induced HCO3 secretion in the absence of butyrate-dependent HCO3 secretion. Therefore, HCO3 secretion was induced in the crypt by the addition of 1 mM 8-Br-cAMP to the serosal solution followed by luminal perfusion of 20 mM butyrate. Figure 4B demonstrates that cAMP-induced HCO3 secretion remained constant during microperfusion with lumen butyrate. In parallel crypt microperfusion studies the effect of butyrate in the serosal solution on cAMP-induced HCO3 secretion was also examined. Addition of 20 mM butyrate in the serosal solution also did not affect cAMP-induced HCO3 secretion. These studies establish that neither lumen nor serosal butyrate downregulated cAMP-induced HCO3 secretion in isolated colonic crypts.

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).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 5. Effect of outward HCO3 concentration ([HCO3]) gradient and butyrate concentration ([butyrate]) gradient on 14C-butyrate uptake in apical membrane vesicles (AMV) from surface and crypt cells. Outward [HCO3] gradient-driven butyrate (butyrate/HCO3 exchange) and [butyrate] gradient-driven butyrate (butyrate/butyrate exchange) uptake was measured in AMV from both surface and crypt cells, as described in MATERIALS AND METHODS. Both butyrate/HCO3 exchange and butyrate/butyrate exchange were present in AMV from surface, but not from crypt cells.

 


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. Effect of outward [butyrate] gradient on 36Cl uptake in AMV from surface and crypt cells. Outward [butyrate] gradient-driven Cl uptake (Cl/butyrate exchange) was measured in AMV from surface and crypt cells, as described in MATERIALS AND METHODS. Cl/butyrate exchange was identified in AMV prepared from both surface and crypt cells.

 
To determine the relative rates of apical membrane uptake of butyrate and isobutyrate, an experiment was performed to compare the initial rate of 14C-butyrate/HCO3 exchange and 14C-isobutyrate/HCO3 exchange in AMV isolated from rat distal colon. The initial rate of [HCO3] gradient-driven 14C-butyrate was 35% higher than that of 14C-isobutyrate uptake (686.3 ± 7.7 vs. 510 ± 38.4 pmol·mg protein–1·3 s–1). These observations indicate that the initial rate of butyrate/HCO3 exchange is greater than that of isobutyrate/HCO3 exchange.

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.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 7. Effect of butyrate on anion exchanges. Cl/HCO3 exchange, Cl/Cl exchange, and Cl/OH exchange in AMV were measured in the presence and absence of 25 mM butyrate, as described in MATERIALS AND METHODS. Extravesicular butyrate almost completely inhibited Cl/HCO3 exchange, whereas Cl/Cl and Cl/OH exchanges were inhibited by 68 and 40%, respectively.

 


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8. Effect of butyrate on anion channel activity. Intravesicular membrane positive potential-dependent electrogenic 36Cl uptake (anion channel activity) in crypt-AMV was measured, as described in MATERIALS AND METHODS. Extravesicular butyrate almost completely inhibited the electrogenic Cl channels.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Short-chain fatty acids have multiple effects on colonic epithelial cell function including both cell differentiation and epithelial ion transport (4, 7, 15, 20, 22, 23, 25, 33). SCFA enhance electroneutral Na-Cl and fluid absorption, which is the basis for the concept that SCFA represent an adaptive mechanism for the conservation of calories, fluid, and electrolytes. Thus the primary mechanism of antibiotic-associated diarrhea that is not due to Clostridium difficile is a reduction in SCFA production by colonic bacteria with a resulting decrease in SCFA stimulation of fluid absorption. The present model of SCFA stimulation of Na-Cl absorption is based on SCFA uptake across the apical cell membrane as a result of SCFA/HCO3 exchange (or via nonionic diffusion) coupled to NHE and Cl/SCFA exchanges (4, 7, 23, 25).

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·h–1·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·h–1·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.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by the Wellcome Trust and National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-60069.


    ACKNOWLEDGMENTS
 
Present address of S. Vidyasagar: Dept. of Medicine, University of Rochester, Rochester, NY.


    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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Argenzio RA, Miller N, and von Engelhardt W. Effect of volatile fatty acids on water and ion absorption from the goat colon. Am J Physiol 229: 997–1002, 1975.[Abstract/Free Full Text]
  2. Argenzio RA and Whipp SC. Inter-relationship of sodium, chloride, bicarbonate and acetate transport by the colon of the pig. J Physiol 295: 365–381, 1979.[Abstract]
  3. Binder HJ and Mehta P. Characterization of butyrate-dependent electroneutral Na-Cl absorption in the rat distal colon. Pflügers Arch 417: 365–369, 1990.[CrossRef][ISI][Medline]
  4. Binder HJ and Mehta P. Short-chain fatty acids stimulate active sodium and chloride absorption in vitro in the rat distal colon. Gastroenterology 96: 989–996, 1989.[ISI][Medline]
  5. Bugaut M. Occurrence, absorption and metabolism of short chain fatty acids in the digestive tract of mammals. Comp Biochem Physiol B 86: 439–472, 1987.[ISI][Medline]
  6. Charney AN, Giannella RA, and Egnor RW. Effect of short-chain fatty acids on cyclic 3',5'-guanosine monophosphate-mediated colonic secretion. Comp Biochem Physiol A Mol Integr Physiol 124: 169–178, 1999.[CrossRef][ISI][Medline]
  7. Charney AN, Micic L, and Egnor RW. Nonionic diffusion of short-chain fatty acids across rat colon. Am J Physiol Gastrointest Liver Physiol 274: G518–G524, 1998.[Abstract/Free Full Text]
  8. Clarke LL and Harline MC. Dual role of CFTR in cAMP-stimulated HCO3 secretion across murine duodenum. Am J Physiol Gastrointest Liver Physiol 274: G718–G726, 1998.[Abstract/Free Full Text]
  9. Cuff MA, Lambert DW, and Shirazi-Beechey SP. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1. J Physiol 539: 361–371, 2002.[Abstract/Free Full Text]
  10. Dagher PC, Egnor RW, Taglietta-Kohlbrecher A, and Charney AN. Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon. Am J Physiol Cell Physiol 271: C1853–C1860, 1996.[Abstract/Free Full Text]
  11. Dietz J and Field M. Ion transport in rabbit ileal mucosa. IV. Bicarbonate secretion. Am J Physiol 225: 858–861, 1973.[Free Full Text]
  12. Feldman GM and Stephenson RL. H+ and HCO3 flux across apical surface of rat distal colon. Am J Physiol Cell Physiol 259: C35–C40, 1990.[Abstract/Free Full Text]
  13. Geibel JP, Singh S, Rajendran VM, and Binder HJ. hco3 secretion in the rat colonic crypt is closely linked to Cl secretion. Gastroenterology 118: 101–107, 2000.[ISI][Medline]
  14. Hadjiagapiou C, Schmidt L, Dudeja PK, Layden TJ, and Ramaswamy K. Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1. Am J Physiol Gastrointest Liver Physiol 279: G775–G780, 2000.[Abstract/Free Full Text]
  15. Heerdt BG, Houston MA, and Augenlicht LH. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines. Cancer Res 54: 3288–3293, 1994.[Abstract]
  16. Hogan DL, Crombie DL, Isenberg JI, Svendsen P, Schaffalitzky de Muckadell OB, and Ainsworth MA. CFTR mediates cAMP- and Ca2+-activated duodenal epithelial HCO3 secretion. Am J Physiol Gastrointest Liver Physiol 272: G872–G878, 1997.[Abstract/Free Full Text]
  17. Illek B, Yankaskas JR, and Machen TE. cAMP and genistein stimulate HCO3 conductance through CFTR in human airway epithelia. Am J Physiol Lung Cell Mol Physiol 272: L752–L761, 1997.[Abstract/Free Full Text]
  18. Krishnan S, Rajendran VM, and Binder HJ. Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon. Am J Physiol Cell Physiol 285: C1246–C1254, 2003.[Abstract/Free Full Text]
  19. Krishnan S, Ramakrishna BS, and Binder HJ. Stimulation of sodium chloride absorption from secreting rat colon by short-chain fatty acids. Dig Dis Sci 44: 1924–1930, 1999.[CrossRef][ISI][Medline]
  20. Kunzelmann K and Mall M. Electrolyte transport in the mammalian colon: mechanisms and implications for disease. Physiol Rev 82: 245–289, 2002.[Abstract/Free Full Text]
  21. Lomax RB, McNicholas CM, Lombes M, and Sandle GI. Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon. Am J Physiol Gastrointest Liver Physiol 266: G71–G82, 1994.[Abstract/Free Full Text]
  22. Mariadason JM, Catto-Smith A, and Gibson PR. Modulation of distal colonic epithelial barrier function by dietary fibre in normal rats. Gut 44: 394–399, 1999.[Abstract/Free Full Text]
  23. Mascolo N, Rajendran VM, and Binder HJ. Mechanism of short-chain fatty acid uptake by apical membrane vesicles of rat distal colon. Gastroenterology 101: 331–338, 1991.[ISI][Medline]
  24. Minhas BS, Sullivan SK, and Field M. Bicarbonate secretion in rabbit ileum: electrogenicity, ion dependence, and effects of cyclic nucleotides. Gastroenterology 105: 1617–1629, 1993.[ISI][Medline]
  25. Rajendran VM and Binder HJ. Apical membrane Cl-butyrate exchange: mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon. J Membr Biol 141: 51–58, 1994.[ISI][Medline]
  26. Rajendran VM and Binder HJ. Distribution and regulation of apical Cl/anion exchanges in surface and crypt cells of rat distal colon. Am J Physiol Gastrointest Liver Physiol 276: G132–G137, 1999.[Abstract/Free Full Text]
  27. Rajendran VM, Kashgarian M, and Binder HJ. Aldosterone induction of electrogenic sodium transport in the apical membrane vesicles of rat distal colon. J Biol Chem 264: 18638–18644, 1989.[Abstract/Free Full Text]
  28. Rajendran VM, Singh SK, Geibel J, and Binder HJ. Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells. Am J Physiol Gastrointest Liver Physiol 274: G424–G429, 1998.[Abstract/Free Full Text]
  29. Reynolds DA, Rajendran VM, and Binder HJ. Bicarbonate-stimulated [14C]butyrate uptake in basolateral membrane vesicles of rat distal colon. Gastroenterology 105: 725–732, 1993.[ISI][Medline]
  30. Ritzhaupt A, Ellis A, Hosie KB, and Shirazi-Beechey SP. The characterization of butyrate transport across pig and human colonic luminal membrane. J Physiol 507: 819–830, 1998.[Abstract/Free Full Text]
  31. Ritzhaupt A, Wood IS, Ellis A, Hosie KB, and Shirazi-Beechey SP. Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate. J Physiol 513: 719–732, 1998.[Abstract/Free Full Text]
  32. Ritzhaupt A, Wood IS, Ellis A, Hosie KB, and Shirazi-Beechey SP. Identification of a monocarboxylate transporter isoform type 1 (MCT1) on the luminal membrane of human and pig colon (Abstract). Biochem Soc Trans 26: S120, 1998.[Medline]
  33. Roediger WE. Utilization of nutrients by isolated epithelial cells of the rat colon. Gastroenterology 83: 424–429, 1982.[ISI][Medline]
  34. Ruppin H, Bar-Meir S, Soergel KH, Wood CM, and Schmitt MG Jr. Absorption of short-chain fatty acids by the colon. Gastroenterology 78: 1500–1507, 1980.[ISI][Medline]
  35. Seidler U, Blumenstein I, Kretz A, Viellard-Baron D, Rossmann H, Colledge WH, Evans M, Ratcliff R, and Gregor M. A functional CFTR protein is required for mouse intestinal cAMP-cGMP- and Ca2+-dependent HCO3 secretion. J Physiol 505: 411–423, 1997.[Abstract]
  36. Sellin JH and Desoignie R. Regulation of bicarbonate transport in rabbit ileum: pH stat studies. Am J Physiol Gastrointest Liver Physiol 257: G607–G615, 1989.[Abstract/Free Full Text]
  37. Singh SK, Binder HJ, Boron WF, and Geibel JP. Fluid absorption in isolated perfused colonic crypts. J Clin Invest 96: 2373–2379, 1995.[ISI][Medline]
  38. Smith PL, Cascairo MA, and Sullivan SK. Sodium dependence of luminal alkalinization by rabbit ileal mucosa. Am J Physiol Gastrointest Liver Physiol 249: G358–G368, 1985.[Abstract/Free Full Text]
  39. Stieger B, Marxer A, and Hauri HP. Isolation of brush-border membranes from rat and rabbit colonocytes: is alkaline phosphatase a marker enzyme? J Membr Biol 91: 19–31, 1986.[ISI][Medline]
  40. Sullivan SK and Smith PL. Bicarbonate secretion by rabbit proximal colon. Am J Physiol Gastrointest Liver Physiol 251: G436–G445, 1986.[Abstract/Free Full Text]
  41. Umesaki Y, Yajima T, Yokokura T, and Mutai M. Effect of organic acid absorption on bicarbonate transport in rat colon. Pflügers Arch 379: 43–47, 1979.[CrossRef][ISI][Medline]
  42. Vidyasagar S, Rajendran VM, and Binder HJ. Three distinct mechanisms of HCO3 secretion in rat distal colon. Am J Physiol Cell Physiol 287: C612–C621, 2004.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/6/G1217    most recent
00415.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Vidyasagar, S.
Articles by Rajendran, V. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Vidyasagar, S.
Articles by Rajendran, V. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.