Nonionic diffusion of short-chain fatty acids across rat colon

Alan N. Charney, Ljubisa Micic, and Richard W. Egnor

Nephrology Section, Veterans Affairs Medical Center, New York University School of Medicine, New York, New York 10010

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Short-chain fatty acid (SCFA) transport across the colon may occur by nonionic diffusion and/or via apical membrane SCFA-/HCO<SUP>−</SUP><SUB>3</SUB> exchange. To examine the relative importance of these processes, stripped segments of rat (Ratus ratus) proximal and distal colon were studied in Ussing chambers, and the unidirectional fluxes of radiolabeled SCFA butyrate, propionate, or weakly metabolized isobutyrate were measured. In N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or 1 or 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer, decreases in mucosal pH stimulated mucosal-to-serosal flux (Jmright-arrow s) of all SCFA, decreases in serosal pH stimulated serosal-to-mucosal flux (Jsright-arrow m), and bilateral pH decreases stimulated both fluxes equally. These effects were observed whether the SCFA was present on one or both sides of the tissue, in both proximal and distal colon, in the absence of luminal Na+, and in the presence of either luminal or serosal ouabain. Changes in intracellular pH or intracellular [HCO<SUP>−</SUP><SUB>3</SUB>] did not account for the effects of extracellular pH. Luminal Cl- removal, to evaluate the role of apical membrane Cl-/SCFA- exchange, had no effect on Jmright-arrow s but decreased Jsright-arrow m 32% at pH 6.5 and 22% at 7.2. Increasing SCFA concentration from 1 to 100 mM, at pH 6.4 or 7.4, caused a linear increase in Jmright-arrow s. We conclude that SCFA are mainly transported across the rat colon by nonionic diffusion.

butyrate; propionate; in vitro; flux; pH; intracellular pH

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SHORT-CHAIN FATTY ACIDS (SCFA) are produced by bacterial metabolism of unabsorbed carbohydrates in the mammalian colon. They provide the predominant anions in the colonic lumen and include acetic (60-75%), propionic (15-25%), and butyric acids (10-15%). Once absorbed, SCFA stimulate Na+ and Cl- absorption, contribute to the maintenance of cell pH and volume, and contribute potential base to the systemic acid-base pool (5). The transport and metabolic pathways by which each of these functions is achieved are well described.

Until recently, the colonic absorption of SCFA was believed to occur through nonionic diffusion. This mechanism of SCFA passage across the luminal and serosal membranes is consistent with the functions noted above and with partial recycling of SCFA across the luminal membrane via Cl-/SCFA- exchange (4, 25). This passive transport process is stimulated by decreases in bulk fluid or microclimate luminal pH consistent with the fact that the acid dissociation constant (pKa) of the most abundant SCFA in the colonic lumen is approximately two pH units lower (6).

Recently, an SCFA-/HCO<SUP>−</SUP><SUB>3</SUB> exchange process was identified in apical brush-border membrane vesicles prepared from the rat colon (24). This process also is stimulated by reductions in luminal pH and exhibits a Michaelis constant (Km) for butyrate of 27 mM, near or below typical SCFA concentrations found in the colonic lumen. It was suggested that at least in this segment of this species, the major mechanism by which SCFA are absorbed is luminal membrane anion exchange (24). This mechanism of SCFA absorption had been suggested for the human ileum (21), and in the absence of an identified exchanger, for the rat jejunum (3) and rabbit and guinea pig proximal colon (18, 30). To examine these possibilities in the rat colon, we studied SCFA transport under in vitro conditions designed to test the functional importance rather than the presence of anion exchange. We systematically examined the effects of altering extracellular and intracellular pH (pHe and pHi, respectively) and examined the tenets of carrier-mediated transport. Our intent was to determine to what extent passive movement of SCFA across the luminal and serosal cell membranes could account for transepithelial transport in rat colon.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Male Sprague-Dawley rats weighing 250-350 g were maintained on a standard diet with free access to water. Under pentobarbital sodium anesthesia (5 mg/100 g body wt), the proximal or distal 10 cm of colon were removed and rinsed with 0.9% saline. The serosa was stripped while the tissue was mounted on a glass rod.

Ion flux measurements. Details of the method were previously described (9, 10). Briefly, tissue pairs were mounted in modified Ussing half-chambers exposing 1.12 cm2 surface area. The transepithelial potential difference (PD) was referenced to the mucosal side. Tissues were studied under short-circuit conditions except for 1-s intervals every 100 s, during which bipolar pulses of 0.5 mV yielded electrical current values that were used to calculate tissue conductance (G). Tissues were paired for ion flux studies on the basis of differences in G no greater than 25%. The short-circuit current (Isc) divided by G yielded the active transport PD.

The fluxes of SCFA were measured by adding 1 µCi 14C-SCFA (10-20 mCi/mM specific activity; NEN, Boston, MA) to the mucosal side of one member of each tissue pair and to the serosal side of the other. Mucosal-to-serosal (Jmright-arrow s) and serosal-to-mucosal (Jsright-arrow m) fluxes were measured over a 16-min period after an initial 30-min equilibration period. Twelve minutes were allowed for each new steady state, and 32 min were allowed for the effect of ouabain. Net flux was calculated as (Jmright-arrow s - Jsright-arrow m).

Solutions and acid-base conditions. The composition of the solutions is shown in Table 1. The N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Ringer solutions (A and F) were gassed with 100% O2, and pH was titrated using 2 M H2SO4 or 1 M NaOH. The HCO<SUP>−</SUP><SUB>3</SUB> solutions (B-E) were gassed with 1% CO2 (PCO2 = 7 mmHg), 3% CO2 (PCO2 = 21 mmHg), 5% CO2 (PCO2 = 35 mmHg), 11% CO2 (PCO2 = 75 mmHg), or 14% CO2 (PCO2 = 95 mmHg) (balance O2) to obtain various pH values. All solutions were maintained at 37°C. The solutions were so designed that after the addition of the salt of an SCFA or gluconic acid, similar final osmolality and, where appropriate, Na+ concentration (always <150 mM) were achieved.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Solutions

In the ion replacement experiments, choline and isethionate replaced Ringer Na+ and Cl-, respectively. SCFA concentration was 25 mM bilaterally in all experiments except in the gradient and transport kinetics studies where 1, 10, 25, 50, or 100 mM Na+ butyrate or Na+ isobutyrate were used on one side and Na+ gluconate on the other. In certain experiments, ouabain (1 mM; Sigma Chemical, St. Louis, MO) was added to either the mucosal or serosal bathing solution.

In several experiments in which butyrate flux was measured, the tops of the fluid reservoirs were sealed and vented through an ethanolamine trap (20).14CO2 was quantitated by liquid scintillation counting to determine the degree to which butyrate was metabolized by the colonic epithelium. We found that ~7% of the butyrate that entered cells during transmucosal passage was metabolized to CO2.

pHi measurements. The method is described in detail elsewhere (9-11, 13). Briefly, a segment of stripped distal colon (described previously) was mounted as a flat sheet over a 1-cm2 hollow ring assembly. It was first incubated in Ringer containing 2 mM DL-dithiothreitol (Sigma), a mucolytic agent, for 10 min. It was then bathed in Ringer containing 9.68 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR) for 40 min. Intracellular cleavage of BCECF-AM by endogenous esterases produces the poorly permeable pH-sensitive dye BCECF. The mounted tissue was then washed three times in fresh Ringer solution (as designated by the experimental protocol) to remove extracellular dye. Fluorescence microscopy localized the dye primarily to surface epithelial cells (9).

A Perkin-Elmer LS-5B spectrofluorometer (South Plainfield, NJ) equipped with a thermoregulated cuvette holder was used. The mounted tissue was placed in a fixed position at the bottom of a 4-ml cuvette (Markson Science, Phoenix, AZ) with the mucosal surface facing the excitation beam at a 45° angle. In the experiments with a pH gradient a modified tissue holder was used (10). It consisted of a plastic divider that spanned two opposite inside corners of the cuvette. Stripped colonic tissue was placed over a plastic ring designed to snugly fit a 9-mm hole in the divider. The divider was snapped over the ring and tissue. Silicone grease was used to provide a water-tight seal between the divider and the cuvette.

When acid-base conditions were altered, all readings were performed after pHi reached a plateau, but no less than 12 min after the acid-base condition was changed. During this time the solution in the cuvette was exchanged rapidly with fresh aliquots every 2 min. When HCO<SUP>−</SUP><SUB>3</SUB> buffers were used the cuvette was tightly closed with a plastic cap to prevent CO2 leakage between solution exchanges. These measurements represent steady-state values because in preliminary experiments they were not found to change for periods up to 40 min. Ratiometric fluorescence measurements were performed in triplicate using excitation wavelengths of 440 and 500 nm in sequence. The emission wavelength was 530 nm. The ratio was computed by dividing the fluorescence intensity at 500 nm by that at 440 nm. Only tissues that maintained total fluorescence above 2× autofluorescence for the duration of the experimental protocol were used. Autofluorescence was automatically subtracted.

pHi calibration was done by the high-K, nigericin method (31). The calibration solution contained (in mmol/l) 21 HEPES, 140 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 µg/ml nigericin (Sigma). A logarithmic regression line for the standard curve was used to accommodate the nonlinearity of fluorescence ratios at very low pH values.

Intracellular HCO<SUP>−</SUP><SUB>3</SUB> concentration ([HCO<SUP>−</SUP><SUB>3</SUB>]i) was computed using the Henderson-Hasselbalch equation and the measured pHi. Intracellular PCO2 was assumed to be equal to the medium PCO2, and the negative log of dissociation (pK ') and CO2 solubility were 6.115 and 0.0306, respectively. Bathing solution pH and PCO2 were measured with a Radiometer BMS 3 Mk 2 system with a PHM 73 acid-base analyzer (London Company, Cleveland, OH).Extracellular [HCO<SUP>−</SUP><SUB>3</SUB>] was computed using the Henderson-Hasselbalch equation as described.

All data are expressed as means ± SE and were compared by paired Student's t-test or analysis of variance (ANOVA). Two-tailed P values < 0.05 were considered significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Effect of pH on butyrate flux in distal colon. Initially the effect of unilateral and bilateral changes in bathing solution pH on butyrate flux in HEPES Ringer were examined. In these experiments butyrate was present at 25 mM on both sides of the tissue. As shown in Fig. 1, at pH 7.38 the net flux of butyrate was -0.1 ± 0.3 µeq · cm-2 · h-1. As mucosal solution pH was decreased in steps from 7.38 to 5.47, Jmright-arrow s increased from 3.4 to 7.2 µeq · cm-2 · h-1 and net absorption was observed (3.5 ± 0.3 µeq · cm-2 · h-1). As shown when the luminal pH was then increased to 7.38, the increase in Jmright-arrow s was completely reversible. Luminal pH changes had no effect on the Jsright-arrow m of butyrate.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of mucosal pH change on unilateral flux of butyrate. Tissue pairs were bathed in HEPES Ringer, and butyrate was present on both sides of the tissue at 25 mM. As mucosal solution pH was decreased, mucosal-to-serosal flux (Jmright-arrow s) increased and net butyrate absorption was observed. When mucosal pH was then increased, the increase in Jmright-arrow s was completely reversible. Changes in mucosal pH did not affect serosal-to-mucosal flux (Jsright-arrow m). Values are means ± SE, n = 4-6. Increments and decrement in Jmright-arrow s are significantly different by ANOVA, P < 0.001.

As shown in Fig. 2, as serosal solution pH was decreased in steps from 7.38 to 5.57 Jsright-arrow m of butyrate increased from 3.4 to 5.4 µeq · cm-2 · h-1. This change caused net butyrate secretion (-2.4 ± 0.5 µeq · cm-2 · h-1). When pH was then increased to 7.38 the increase in Jsright-arrow m was completely reversible. Serosal pH changes had no effect on the Jmright-arrow s of butyrate.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of serosal pH change on unilateral flux of butyrate. Tissue pairs were bathed in HEPES Ringer and butyrate was present on both sides of the tissue at 25 mM. As serosal solution pH was decreased, Jsright-arrow m increased and net butyrate secretion was observed. When serosal pH was then increased, the increase in Jsright-arrow m was completely reversible. Changes in serosal pH did not affect Jmright-arrow s. Values are means ± SE, n = 3-5. Increments and decrement in Jsright-arrow m are significantly different by ANOVA, P < 0.01.

The effect of changing the pH of both bathing solutions on butyrate flux is shown in Fig. 3. As solution pH was decreased in steps from 7.39 to 5.53, both Jmright-arrow s and Jsright-arrow m of butyrate increased from ~3 to 7 µeq · cm-2 · h-1. Net butyrate flux was minimal at bilateral pH 7.39 (-0.8 ± 0.4 µeq · cm-2 · h-1) and remained minimal at pH 5.53 (0.1 ± 0.5 µeq · cm-2 · h-1). When pH was then increased to 7.39, the increases in the unidirectional fluxes were completely reversible.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of bilateral pH change on unilateral flux of butyrate. Tissue pairs were bathed in HEPES Ringer and butyrate was present on both sides of the tissue at 25 mM. Net butyrate flux was minimal at pH 7.39. As mucosal and serosal solution pH was decreased, both Jmright-arrow s and Jsright-arrow m increased and net butyrate flux remained minimal. When solution pH was then increased, the increases in Jmright-arrow s and Jsright-arrow m were completely reversible. Values are means ± SE, n = 3-5. Increments and decrement in Jmright-arrow s and Jsright-arrow m are significantly different by ANOVA, P < 0.001.

Reductions in pH of one or both bathing solutions had no effect on G but decreased Isc. For example, when mucosal solution pH was reduced from 7.38 to 5.47, Isc decreased from 0.7 ± 0.2 to 0.2 ± 0.3 µeq · cm-2 · h-1, P < 0.05. When pH was then increased to 7.38 Isc increased to 0.9 ± 0.4 µeq · cm-2 · h-1.

Effect of pH on propionate flux in distal colon. Similar effects of pH changes on propionate fluxes in HEPES Ringer were observed. Propionate was present at 25 mM on both sides of the tissue. Decreases in luminal pH in steps from 7.36 to 5.46 selectively increased Jmright-arrow s of propionate from 2.7 ± 0.2 to 6.3 ± 0.4 µeq · cm-2 · h-1, n = 4, P < 0.001. As serosal solution pH was decreased in steps from 7.36 to 5.46, Jsright-arrow m selectively increased from 4.1 ± 0.2 to 5.4 ± 0.5 µeq · cm-2 · h-1, n = 4, P < 0.05. Minimal net propionate secretion was observed at bilateral pH 7.36 (-1.4 ± 0.3 µeq · cm-2 · h-1, P < 0.02), and zero net transport was found at pH 5.46 (0.9 ± 0.4 µeq · cm-2 · h-1, P < 0.01 compared with flux at pH 7.36).

Effect of butyrate gradient on butyrate flux. We then examined the effects of unilateral pH changes in HEPES Ringer on butyrate fluxes in the presence of a 25 mM luminal to 0 mM serosal butyrate concentration gradient. As mucosal solution pH was decreased in steps from 7.39 to 5.58, Jmright-arrow s increased from 1.0 ± 0.1 to 2.4 ± 0.2 µeq · cm-2 · h-1, n = 4, P < 0.005. When pH was then increased to 7.39, the increase in Jmright-arrow s was reversed to 1.1 ± 0.1 µeq · cm-2 · h-1, P < 0.005. When serosal pH changes were studied in the presence of a 25 mM serosal to 0 mM luminal butyrate concentration gradient, similar results were obtained. As serosal solution pH was decreased in steps from 7.39 to 5.56, Jsright-arrow m of butyrate increased from 2.0 ± 0.1 to 4.1 ± 0.1 µeq · cm-2 · h-1, n = 4, P < 0.007. When pH was then increased to 7.39, the increase in Jsright-arrow m was reversed to 2.3 ± 0.1 µeq · cm-2 · h-1, P < 0.007.

Effect of pH on butyrate flux in HCO<SUP>−</SUP><SUB>3</SUB> Ringer. The effect of bilateral changes in bathing solution pH on butyrate flux across distal colon also was examined in HCO<SUP>−</SUP><SUB>3</SUB> Ringer where pH changes were induced by changing PCO2. In these experiments butyrate was present at 25 mM on both sides of the tissue. In 1 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer at PCO2 = 7 mmHg, pH 6.79, the net flux of butyrate was 0.1 ± 0.2 µeq · cm-2 · h-1, n = 6. As solution pH was decreased in steps to 6.08 by increasing PCO2 to 95 mmHg, net flux was little changed: -0.5 ± 0.2 µeq · cm-2 · h-1. Jmright-arrow s increased from 4.2 ± 0.2 to 4.8 ± 0.3 µeq · cm-2 · h-1 and Jsright-arrow m increased from 4.1 ± 0.3 to 5.3 ± 0.5 µeq · cm-2 · h-1, n = 6, P < 0.05.

In 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer at PCO2 = 7 mmHg, pH 7.24, minimal net butyrate secretion was observed (-1.0 ± 0.2 µeq · cm-2 · h-1, n = 5). As solution pH was decreased to 6.44 in steps by increasing PCO2 to 95 mmHg, net flux was little changed: -0.4 ± 0.5 µeq · cm-2 · h-1. Jmright-arrow s increased from 2.6 ± 0.1 to 4.0 ± 0.3 µeq · cm-2 · h-1 and Jsright-arrow m increased from 3.6 ± 0.2 to 4.4 ± 0.2 µeq · cm-2 · h-1, n = 5, P < 0.02. These flux changes in 1 and 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer were completely reversible and were similar in magnitude to flux changes in HEPES Ringer (~1 µeq · cm-2 · h-1 per pH unit). In addition, in both 1 and 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer, reductions in pH did not affect G but reduced Isc from 0.4 µeq · cm-2 · h-1 to near zero.

Effect of butyrate metabolism on butyrate flux. We then examined whether the metabolism of SCFA influenced the pattern of their transepithelial transport. We studied the effect of pH in HEPES Ringer on the flux across distal colon of isobutyrate, a weakly metabolized SCFA (22). Isobutyrate was present at 25 mM on both sides of the tissue. Decreases in luminal pH in steps from 7.35 to 5.45 increased Jmright-arrow s of isobutyrate from 2.1 ± 0.1 to 4.9 ± 0.1 µeq · cm-2 · h-1, n = 4, P < 0.0001. Jsright-arrow m flux increased slightly from 2.2 ± 0.1 to 2.8 ± 0.1 µeq · cm-2 · h-1, n = 4, P < 0.04. In a separate experiment, when pH was reduced in both bathing solutions in steps, net isobutyrate flux remained unchanged: -1.0 µeq · cm-2 · h-1 at pH 7.34, 0.0 µeq · cm-2 · h-1 at pH 6.68, -0.8 µeq · cm-2 · h-1 at pH 6.04, and -0.5 µeq · cm-2 · h-1 at pH 5.55. These changes in unidirectional fluxes were completely reversible.

Effect of luminal Na+ removal and ouabain. To determine whether apical Na+/H+ exchange activity was necessary for SCFA transport, the exchanger was inhibited by substituting choline for Na+ in the mucosal bathing solution. In HEPES Ringer, with butyrate present at 25 mM on both sides of the tissue, bilateral reductions in pH in steps stimulated Jmright-arrow s and Jsright-arrow m of butyrate equivalently. At pH 7.41 and 5.68, net flux was unchanged and near zero, and Jmright-arrow s was 1.3 ± 0.1 and 5.1 ± 0.1 µeq · cm-2 · h-1 and Jsright-arrow m was 2.1 ± 0.2 and 4.1 ± 0.1 µeq · cm-2 · h-1, respectively, n = 2, P < 0.05. In 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer, similar results were obtained. At pH 7.32 and 6.51, net fluxes were unchanged and near zero, and Jmright-arrow s was 1.5 ± 0.1 and 2.1 ± 0.1 µeq · cm-2 · h-1 and Jsright-arrow m was 2.7 ± 0.1 and 3.4 ± 0.3 µeq · cm-2 · h-1, respectively, n = 3, P < 0.05. The effects of pH in both HEPES and HCO<SUP>−</SUP><SUB>3</SUB> Ringer were completely reversible.

The effect of luminal ouabain was tested to determine whether an apical membrane H+-K+-adenosinetriphosphatase (ATPase) participated in the action of luminal pH on SCFA absorption. The experiments were carried out in 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer with butyrate at 25 mM on both sides of the tissue. Ouabain (1 mM) did not affect Jmright-arrow s at pH 7.30 (2.7 ± 0.3 vs. 2.6 ± 0.3 µeq · cm-2 · h-1, n = 6). Luminal ouabain also did not affect the stimulatory action of a luminal pH reduction to 6.36 on Jmright-arrow s (3.2 ± 0.5 µeq · cm-2 · h-1, n = 6, P < 0.02).

The effect of serosal ouabain was tested to determine if any active transport process was involved in the SCFA response to pH. Fluxes were not measured for 32 min and/or until the Isc was reduced to near zero. The experiments were carried out in 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer with butyrate at 25 mM on both sides of the tissue. As shown in Fig. 4, the addition of 1 mM ouabain to the serosal solution did not alter butyrate fluxes. When pH was reduced on both sides of the tissue from 7.29 to 6.45, increases in both Jmright-arrow s (3.0 ± 0.2 vs. 4.4 ± 0.2 µeq · cm-2 · h-1, n = 4, P < 0.01) and Jsright-arrow m (3.3 ± 0.2 vs. 4.0 ± 0.1 µeq · cm-2 · h-1, n = 4, P < 0.05) were noted.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of serosal ouabain on unilateral flux of butyrate. Tissue pairs were bathed in 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer and butyrate was present at 25 mM on both sides of the tissue. Serosal addition of 1 mM ouabain did not alter butyrate flux at pH 7.29 or affect the stimulation of Jmright-arrow s or Jsright-arrow m when pH was reduced to 6.45 on both sides of the tissue (P < 0.01 and P < 0.05, respectively, by paired Student's t-test). Values are means ± SE, n = 4.

Effect of luminal Cl- removal on butyrate flux. To evaluate the role of apical membrane Cl-/SCFA- exchange, we examined the effect of substituting isethionate for Cl- in the mucosal bathing solution. In 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer, the absence of luminal Cl- at pH 7.21 did not significantly increase Jmright-arrow s of butyrate [2.6 ± 0.1 vs. 3.2 ± 0.3 µeq · cm-2 · h-1, n = 5, not significant (NS)] but did decrease Jsright-arrow m 22% (3.6 ± 0.2 vs. 2.4 ± 0.2 µeq · cm-2 · h-1, n = 5, P < 0.05). At pH 6.50, the removal of luminal Cl- did not increase Jmright-arrow s (4.0 ± 0.3 vs. 4.7 ± 0.5 µeq · cm-2 · h-1, n = 5, NS) and again decreased Jsright-arrow m ~32% (4.4 ± 0.2 vs. 3.0 ± 0.3 µeq · cm-2 · h-1, n = 5, P < 0.005). The absence of luminal Cl- also altered the effect of pH on butyrate flux. A reduction in pH from 7.21 to 6.50 stimulated net flux from 0.8 ± 0.2 to 1.7 ± 0.4 µeq · cm-2 · h-1, n = 5, P < 0.01, as a consequence of a greater increase in Jmright-arrow s (3.2 ± 0.3 vs. 4.7 ± 0.5 µeq · cm-2 · h-1, P < 0.01) than in Jsright-arrow m (2.4 ± 0.2 vs. 3.0 ± 0.3 µeq · cm-2 · h-1, P < 0.02). Cl- removal had similar effects on fluxes in HEPES Ringer, and the effects of pH in both HCO<SUP>−</SUP><SUB>3</SUB> and HEPES Ringer were completely reversible.

Effect of SCFA concentration. Carrier-mediated transport processes, unlike nonionic diffusion, exhibit saturation as evidenced by flattening of the curve describing the relationship between substrate concentration and flux. We examined for saturation by measuring Jmright-arrow s of butyrate in HEPES or 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer at pH 6.40 or 7.40 in the presence of a mucosal-to-serosal butyrate gradient. As shown in Fig. 5, when the mucosal butyrate concentration was progressively increased from 1 to 100 mM, regardless of the Ringer or pH, Jmright-arrow s increased in a linear fashion. Furthermore, the weakly metabolized SCFA isobutyrate exhibited similar transport behavior as its concentration was increased in HCO<SUP>−</SUP><SUB>3</SUB> Ringer at pH 6.40. 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of short-chain fatty acid (SCFA) concentration on Jmright-arrow s of butyrate and isobutyrate. SCFA concentration was increased from 1 to 100 mM in HEPES or 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer at pH 6.40 or 7.40 in presence of a mucosal-to-serosal butyrate gradient. When mucosal SCFA concentration was increased, regardless of Ringer or pH, Jmright-arrow s increased in a linear fashion and saturation kinetics were not observed.

Effect of pHi and [HCO<SUP>−</SUP><SUB>3</SUB>]i. To determine whether the relation of SCFA flux to solution pH changes could be accounted for by changes in intracellular acid-base conditions, we measured pHi and calculated [HCO<SUP>−</SUP><SUB>3</SUB>]i in distal colon during the various experimental conditions. As shown in Table 2, in HEPES Ringer containing 25 mM butyrate and gassed with 100% O2, CO2 tension and therefore [HCO<SUP>−</SUP><SUB>3</SUB>]i were zero. pHi mirrored pHe whether the pH change was unilateral or bilateral. However, when the pHe decrease was unilateral, the decrease in pHi was less than when the pHe change was bilateral. Furthermore, mucosal changes in pHe seemed to have a somewhat greater effect on pHi than serosal changes.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of pHe on colonic pHi in HEPES Ringer

In 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer containing 25 mM butyrate, qualitatively similar effects of bilateral changes in pH were observed. Increasing bathing solution PCO2 from 7 mmHg (pHe 7.43 ± 0.02) to 95 mmHg (pHe 6.48 ± 0.01) decreased pHi from 7.39 ± 0.02 to 6.46 ± 0.03, n = 6, P < 0.001, and increased [HCO<SUP>−</SUP><SUB>3</SUB>]i from 4.8 ± 0.5 to 5.8 ± 0.4 mM, n = 6, P < 0.05. In 5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer containing 25 mM isobutyrate, similar effects of PCO2 on pHi and [HCO<SUP>−</SUP><SUB>3</SUB>]i were observed. When bilateral pHe was decreased from 7.44 to 6.48, pHi decreased from 7.20 ± 0.02 to 6.53 ± 0.02 and [HCO<SUP>−</SUP><SUB>3</SUB>]i increased from 3.0 ± 0.1 to 7.1 ± 0.3 mM, n = 5, P < 0.001. In both HEPES and HCO<SUP>−</SUP><SUB>3</SUB> Ringer, the presence of unilateral or bilateral SCFA did not affect the steady-state value of pHi.

Effect of pH on butyrate flux in proximal colon. We then examined whether the pattern of pH effects on SCFA transport was similar in the proximal colon. Butyrate flux was measured when present at 25 mM on both sides of the tissue in HEPES Ringer. Bilateral decreases in pH from 7.39 to 5.69 in steps increased Jmright-arrow s from 2.9 ± 0.2 to 4.5 ± 0.3 µeq · cm-2 · h-1, n = 4, P < 0.01, and Jsright-arrow m from 2.2 ± 0.4 to 3.3 ± 0.4 µeq · cm-2 · h-1, n = 4, P < 0.002. Net butyrate fluxes were minimal at bilateral pH 7.39 (0.8 ± 0.5 µeq · cm-2 · h-1) and pH 5.69 (1.2 ± 0.5 µeq · cm-2 · h-1, n = 4, NS). Reductions in pH also had no effect on G but decreased Isc. All of these changes were qualitatively and quantitatively similar to those observed in distal colon.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The importance of SCFA in colonic energy metabolism (5, 26), ion transport (1, 2, 4, 12, 17, 26, 28, 32), pHi regulation (7, 14, 15), and systemic acid-base balance (5) has been recognized for some time. SCFA absorption precedes and is required for these functions. For many years the mechanism of SCFA absorption by the colon was believed to be by nonionic diffusion. The basis for this were the findings that chain length, luminal pH, and the concentration gradient from lumen to blood (or tissue or serosa) affected the absorption rate (5, 29, and see Ref. 30 for a review of these considerations). However, such characteristics are compatible with SCFA absorption through apical and basolateral membrane SCFA-/HCO<SUP>−</SUP><SUB>3</SUB> exchange processes. Indeed, these process have recently been identified in colonic membrane vesicles (20, 24, 27).

Our experiments were designed to determine the relative importance of nonionic diffusion and anion exchange as mechanisms by which SCFA cross the colonic mucosa. We considered the Km of the apical anion exchanger (27 mM for butyrate in rat colon) (24), the pKa of the SCFA under consideration (4.8-4.9), metabolism of SCFA by the colonic mucosa, the cell-to-lumen flux of SCFA by apical Cl-/SCFA- exchange (25), and the possibility that various SCFA or segments of the colon had different transport characteristics (30). Thus experiments were performed at unilateral or bilateral butyrate concentrations of 25 mM; with butyrate, propionate, and weakly metabolized isobutyrate (22); over a pH range of 7.4 to 5.3; and in the presence and absence of luminal Na+ or Cl-, or mucosal or serosal ouabain. Our findings strongly suggest that a passive transport pathway, presumably nonionic diffusion, accounts for SCFA transport across rat colon.

The most compelling evidence for nonionic diffusion includes the findings that unilateral reductions in pHe had selective and quantitatively equivalent effects on Jmright-arrow s and Jsright-arrow m, that the effects of pHe were similar in HEPES and HCO<SUP>−</SUP><SUB>3</SUB> Ringer, and that transepithelial transport was unsaturable at SCFA concentrations up to 100 mM. None of these findings would be expected or accounted for by a carrier-mediated epithelial transport process and by apical membrane SCFA-/HCO<SUP>−</SUP><SUB>3</SUB> exchange in particular. It is otherwise difficult to explain how SCFA could move at equivalent rates in both directions across colonic tissue if the transport process were not passive, how SCFA could traverse the tissue via a SCFA-/HCO<SUP>−</SUP><SUB>3</SUB> exchange process in the apparent absence of intracellular bicarbonate (in HEPES Ringer), and why transport saturation would not be observed at substrate concentrations almost four times greater than the Km (observed in brush-border membrane vesicles).

The measurements of pHi also shed light on the mechanism of SCFA transport. In HEPES Ringer, the effects of pHe on unidirectional fluxes were equivalent whether the changes in pHe were unilateral or bilateral. Moreover, the absolute values for the unidirectional fluxes were equivalent at similar values for unilateral pHe and bilateral pHe. However, in HEPES Ringer (Table 2), the effects of unilateral and bilateral changes in pHe on pHi were not equivalent. This suggests that the effect of pHe on SCFA flux was primarily localized to the mucosal or serosal compartment in which it occurred rather than through the effects of pHe on pHi. As discussed previously, such an effect would be more compatible with the transport process of nonionic diffusion than anion exchange.

We also found that luminal removal of Na+ to inhibit apical Na+/H+ exchange did not alter SCFA transport in rat colon or the effects of pHe. Furthermore, neither luminal ouabain, which may inhibit colonic apical membrane H+-K+-ATPase (8, 16, 19, 23), nor serosal ouabain, which inhibits all active transport processes, affected SCFA transport or the effects of pHe. Epinephrine stimulation of apical Na+/H+ exchange has been shown to stimulate propionate absorption in rabbit proximal but not distal colon (29, 30). A pH gradient (presumably luminal microclimate pHe < pHi) was suggested as the mechanism of these effects (29). Because we could not confirm a role for apical Na+/H+ exchange in SCFA absorption, we believe that the reported requirement for this exchanger may be species specific. The presence of a microclimate pH, of course, is consistent with both SCFA absorption by nonionic diffusion and anion exchange.

Metabolism of SCFA by the colonic mucosa certainly occurs, and in preliminary experiments we found that ~7% of the butyrate that entered cells was metabolized to CO2. In the rabbit proximal colon in vitro, from 4 to 7% of absorbed propionate was metabolized to CO2 under similar experimental conditions (29). We do not believe that SCFA metabolism affected our flux measurements or their interpretation. First, in our studies the absolute fluxes and the effects of pH and substrate concentration were similar for butyrate, propionate, and weakly metabolized isobutyrate. Second, the presence of glucose in our studies reduced the fraction of colonic energy derived from SCFA (5). Third, our unidirectional flux calculations were based on the appearance of radiolabeled butyrate in the unlabeled "cold" bathing solution rather than on the disappearance of butyrate from the labeled "hot" side. Thus it is irrelevant to the calculation of unidirectional flux that ~7% more butyrate entered cells than exited into the opposite bathing solution.

In addition to transport across the cell, SCFA may be recycled across the apical membrane. Recycling presumably occurs via the Cl-/SCFA- exchange process described by Rajendran and Binder (25). In our studies of rat colon, the fraction recycled was estimated by comparing the Jmright-arrow s of butyrate in the presence and absence of luminal Cl-. We found that Jmright-arrow s was not significantly affected by the absence of this anion. Jsright-arrow m, however, was decreased 22-32%, depending on pHe. Apparently, under the experimental conditions described (5 mM HCO<SUP>−</SUP><SUB>3</SUB> Ringer at pHe between 6.50 and 7.21) apical membrane Cl-/SCFA- exchange has a greater role in the transcellular secretory flux of SCFA than in apical membrane recycling of absorbed SCFA.

Our findings do not rule out a contribution of anion exchange at the apical and basolateral membranes to SCFA absorption in situ. The effects on SCFA transport of intact tissue layers, blood flow, cell membrane potential, competing substrates, and varying energy stores and demands are unknown. Moreover, the complexity of the in situ environment suggests that the relative importance of active and passive transport of SCFA may not be fixed. Nevertheless, the experimental conditions examined here do mirror in situ conditions where the [HCO<SUP>−</SUP><SUB>3</SUB>]i is very low and a lumen-to-blood pH gradient and SCFA concentration gradient exist. Indeed, such conditions favor net absorption of SCFA by nonionic diffusion. Our studies suggest that at least in the rat colon nonionic diffusion is the most important if not the only mechanism of SCFA absorption.

    ACKNOWLEDGEMENTS

The authors appreciate the technical assistance of Matthew Jenkins.

    FOOTNOTES

This material is based on work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.

Address for reprint requests: A. N. Charney, Nephrology Section, VA Medical Center, 423 East 23rd St., New York, NY 10010.

Received 19 August 1997; accepted in final form 3 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Argenzio, R. A., M. Southworth, J. E. Lowe, and C. E. Stevens. Interrelationship of Na, HCO3, and volatile fatty acid transport by equine large intestine. Am. J. Physiol. 233 (Endocrinol. Metab. Gastrointest. Physiol. 2): E469-E478, 1977[Medline].

2.   Argenzio, R. A., and S. C. Whipp. Interrelationship of sodium, chloride, bicarbonate and acetate transport by the colon of the pig. J. Physiol. (Lond.) 295: 365-381, 1979[Abstract].

3.   Barry, R. J. C., M. J. Jackson, and D. H. Smyth. Transfer of propionate by rat small intestine in vitro. J. Physiol. (Lond.) 182: 150-163, 1966[Medline].

4.   Binder, H. J., and P. Mehta. Short-chain fatty acids stimulate active sodium and chloride absorption in vitro in the rat distal colon. Gastroenterology 96: 989-996, 1989[Medline].

5.   Bugaut, M. Occurrence, absorption and metabolism of short-chain fatty acids in the digestive tract of mammals. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 86: 439-472, 1987.

6.   Chu, S., and M. H. Montrose. Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids. Proc. Natl. Acad. Sci. USA 92: 3303-3307, 1995[Abstract].

7.   Chu, S., and M. H. Montrose. An Na+-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes. J. Gen. Physiol. 105: 589-615, 1995[Abstract].

8.   Cougnon, M., G. Planelles, M. S. Crowson, G. E. Shull, B. C. Rossier, and F. Jaisser. The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase. J. Clin. Invest. 96: 2002-2008, 1995[Medline].

9.   Dagher, P. C., L. Balsam, J. T. Weber, R. W. Egnor, and A. N. Charney. Modulation of chloride secretion in the rat colon by intracellular bicarbonate. Gastroenterology 103: 120-127, 1992[Medline].

10.   Dagher, P. C., T. Behm, A. Taglietta-Kohlbrecher, R. W. Egnor, and A. N. Charney. Dissociation of colonic apical Na/H exchange activity from bulk cytoplasmic pH. Am. J. Physiol. 270 (Cell Physiol. 39): C1799-C1806, 1996[Abstract/Free Full Text].

11.   Dagher, P. C., R. W. Egnor, and A. N. Charney. Effect of intracellular acidification on colonic NaCl absorption. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G569-G575, 1993[Abstract/Free Full Text].

12.   Dagher, P. C., R. W. Egnor, A. Taglietta-Kohlbrecher, and A. N. Charney. Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon. Am. J. Physiol. 271 (Cell Physiol. 40): C1853-C1860, 1996[Abstract/Free Full Text].

13.   Dagher, P. C., T. Z. Morton, C. J. Joo, A. Taglietta-Kohlbrecher, R. W. Egnor, and A. N. Charney. Modulation of secretagogue-induced chloride secretion by intracellular bicarbonate. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G929-G934, 1994[Abstract/Free Full Text].

14.   DeSoignie, R., and J. H. Sellin. Propionate-initiated changes in intracellular pH in rabbit colonocytes. Gastroenterology 107: 347-356, 1994[Medline].

15.   Diener, M., C. Helmle-Kolb, H. Murer, and E. Scharrer. Effect of short-chain fatty acids on cell volume and intracellular pH in rat distal colon. Pflügers Arch. 424: 216-223, 1993[Medline].

16.   Diener, M., F. Hug, D. Strabel, and E. Scharrer. Cyclic AMP-dependent regulation of K+ transport in the rat distal colon. Br. J. Pharmacol. 118: 1477-1487, 1996[Abstract].

17.   Diener, M., A. Peter, and E. Scharrer. The role of volume-sensitive Cl- channels in the stimulation of chloride absorption by short-chain fatty acids in the rat colon. Acta Physiol. Scand. 151: 385-394, 1994[Medline].

18.   Engelhardt, W. V., G. Gros, M. Burmester, K. Hansen, G. Becker, and G. Rechkemmer. Functional role of bicarbonate in propionate transport across guinea-pig isolated caecum and proximal colon. J. Physiol. (Lond.) 477: 365-371, 1994[Abstract].

19.   Feldman, G. M., and J. W. Ickes. Net H+ and K+ fluxes across the apical surface of rat distal colon. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G54-G62, 1997[Abstract/Free Full Text].

20.   Harig, J. M., E. K. Ng, P. K. Dudeja, T. A. Brasitus, and K. Ramaswamy. Transport of n-butyrate into human colonic luminal membrane vesicles. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G415-G422, 1996[Abstract/Free Full Text].

21.   Harig, J. M., K. H. Soergel, J. A. Barry, and K. Ramaswamy. Transport of propionate by human ileal brush-border membrane vesicles. Am. J. Physiol. 260 (Gastrointest. Liver Physiol. 23): G776-G782, 1991[Abstract/Free Full Text].

22.   Jaskiewicz, J., Y. Zhao, J. W. Hawes, Y. Shimomura, D. W. Crabb, and R. A. Harris. Catabolism of isobutyrate by colonocytes. Arch. Biochem. Biophys. 327: 265-270, 1996[Medline].

23.   Lee, J., V. M. Rajendran, A. S. Mann, M. Kashgarian, and H. J. Binder. Functional expression and segmental localization of rat colonic K-adenosine triphosphatase. J. Clin. Invest. 96: 2002-2008, 1995[Medline].

24.   Mascolo, N., V. M. Rajendran, and H. J. Binder. Mechanism of short-chain fatty acid uptake by apical membrane vesicles of rat distal colon. Gastroenterology 101: 331-338, 1991[Medline].

25.   Rajendran, V. M., and H. J. Binder. 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[Medline].

26.   Ramakrishna, B. S., S. H. Nance, I. C. Roberts-Thomson, and W. E. W. Roediger. The effects of enterotoxins and short-chain fatty acids on water and electrolyte fluxes in ileal and colonic loops in vivo in the rat. Digestion 45: 93-101, 1990[Medline].

27.   Reynolds, D. A., V. M. Rajendran, and H. J. Binder. Bicarbonate-stimulated [14C]butyrate uptake in basolateral membrane vesicles of rat distal colon. Gastroenterology 105: 725-732, 1993[Medline].

28.   Ruppin, H., S. Bar-Meir, K. H. Soergel, C. M. Wood, and M. G. Schmitt. Absorption of short-chain fatty acids by the colon. Gastroenterology 78: 1500-1507, 1980[Medline].

29.   Sellin, J. H., and R. DeSoignie. Short-chain fatty acid absorption in rabbit colon in vitro. Gastroenterology 99: 676-683, 1990[Medline].

30.   Sellin, J. H., R. DeSoignie, and S. Burlingame. Segmental differences in short-chain fatty acid transport in rabbit colon: effect of pH and Na. J. Membr. Biol. 136: 147-158, 1993[Medline].

31.   Thomas, J. A., R. N. Buchsbaum, A. Simniak, and E. Racker. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18: 2210-2218, 1979[Medline].

32.   Umesaki, Y., T. Yajima, T. Yokokura, and M. Masahiko. Effect of organic acid absorption on bicarbonate tranport in rat colon. Pflügers Arch. 379: 43-47, 1979[Medline].


AJP Gastroint Liver Physiol 274(3):G518-G524