Department of Medicine, University of Illinois at Chicago and Westside Veterans Administration Medical Center, Chicago, Illinois 60612
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ABSTRACT |
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Current
studies were undertaken to characterize the mechanism of short-chain
fatty acid (SCFA) transport in isolated human proximal colonic
basolateral membrane vesicles (BLMV) utilizing a rapid-filtration
n-[14C]butyrate uptake technique. Human colonic tissues
were obtained from mucosal scrapings from organ donor proximal colons.
Our results, consistent with the existence of a
HCO1 · 5 s
1; 5)
n-[14C]butyrate influx into the vesicles demonstrated a
transstimulation phenomenon; and 6) intravesicular or
extravesicular Cl
did not alter the anion-stimulated
n-[14C]butyrate uptake. Our results indicate the presence
of a carrier-mediated HCO
short-chain fatty acids; transport; human large intestine; contraluminal membranes
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INTRODUCTION |
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SHORT-CHAIN FATTY ACIDS (SCFAs; i.e., acetate, propionate, and butyrate) are the major anions in the colonic lumen produced by anaerobic fermentation of dietary fiber by luminal microflora (4, 6, 8-10). SCFAs are a preferred source of energy for the normal colonic epithelial cells (6-8, 10, 11, 32, 33). SCFAs, particularly butyrate, have been shown to regulate colonic fluid and electrolyte absorption (1, 34, 36), induce cell differentiation, and promote growth and proliferation of the colonic mucosa (8, 25, 39). Variations in the usual colonic production and malabsorption of SCFAs have also been shown to be the potentiating factors in the inflammatory bowel diseases in humans (3, 13, 17, 27, 28). Despite the significance of SCFAs in maintaining the human colonic physiology, the mechanism of SCFA absorption and regulation is still not fully understood.
A number of previous studies using in vivo perfusion experiments (18, 24), flux studies with whole epithelial preparations (5, 19, 43), or isolated membrane vesicles (15, 16, 22, 38) have attempted to characterize the mechanisms of SCFA absorption across the colonic luminal membranes. To date, however, the two main mechanisms proposed for SCFA absorption across the intestinal mucosa are nonionic diffusion of protonated SCFAs produced in the acidic microenvironment of the colonic lumen (5, 37, 42) and/or a carrier-mediated anion exchange (22, 29, 43). It has been suggested that for the nonionic diffusion of SCFAs across the colonic apical membranes, protons may be available from the Na+/H+ exchanger in the apical domains (12) or generated from hydration of CO2 by carbonic anhydrase activity (35). In this regard, previous perfusion studies have shown SCFA absorption to stimulate Na+ uptake (1, 23, 34, 35) and also luminal Na+ concentrations to increase SCFA uptake (41). However, at the physiological colonic luminal pH, the predominant members of SCFA (pKa ~4.8) are in the ionized state and not available for simple diffusion and, therefore, may be transported by a possible carrier-mediated process.
Recent studies from our laboratory and others utilizing vesicle
transport techniques have clearly demonstrated the presence of a
bicarbonate-dependent, carrier-mediated anion exchange mechanism for
SCFA uptake in the apical membranes of the human ileum and colon
(15, 16), rat distal colon (22), and rabbit
intestine (21). In this regard, it has been shown that the
absorbed butyrate is not completely metabolized by the colonocytes
(31-33) and could be further transported across the
colonic basolateral membranes into the blood stream. A recent study by
Reynolds et. al. (30) characterized a distinct
carrier-mediated HCO-gradient-dependent anion exchange in basolateral
membrane vesicles (BLMV) of rat distal colon. The present basolateral
membrane studies were, therefore, undertaken to examine the mechanism
of SCFA uptake in isolated BLMVs of the human proximal colon.
Results of our present study provide strong evidence for the existence
of a carrier-mediated, HCO-HCO
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MATERIALS AND METHODS |
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Materials. SITS, DIDS, amiloride, niflumic acid, and acetazolamide were obtained from Sigma (St. Louis, MO). All other materials were obtained from either Fisher Scientific (Fairlawn, NJ) or Sigma unless otherwise stated and were of highest purity available. Radionuclide n-[1-14C]butyrate (19.7 mCi/mmol) was obtained from New England Nuclear Life Science Products (Boston, MA).
Isolation of human proximal colonic BLMVs.
Approval of the present investigations was obtained from the
Institutional Review Board of the University of Illinois at Chicago. Colons from healthy (primarily trauma victims) adult organ donors (which were declared brain dead) were obtained immediately after harvest of transplantation organs. After the cecum was discarded, the
remaining large intestine was cleaned with an ice-cold 0.9% NaCl and
divided into two equal parts, proximal and distal. The mucosa was
scraped from the seromuscular layer of the proximal colon and stored at
70°C. The human colonic basolateral plasma membranes were purified
utilizing a Percol density gradient technique recently established in
our laboratory (40). The final membrane pellet was
suspended in the transport buffer by passing it 10 times through a
25-gauge (1.5 in.) needle. After the final suspension, the vesicles are
used for uptake studies either within 1-2 h of purification or
frozen at
80°C for later use within a week. Membrane protein was
assessed as described by Bradford (2), using bovine plasma
gamma globulin as standard.
n-[14C]butyrate uptake.
Uptake of n-[14C]butyrate was measured at 25°C by a
rapid filtration technique as described previously (15,
16). Unless otherwise stated, the membrane vesicles were
preloaded with 150 mM KHCO
Statistical analysis. All experiments were performed using at least three to four separate membrane preparations from colons of different organ donors. Paired or unpaired Student's t-tests were used in statistical analysis as appropriate. Results are expressed as means ± SE. A P value < 0.05 was considered statistically significant.
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RESULTS |
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Effect of HCO and HCO
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Effect of transmembrane potential on n-[14C]butyrate
uptake.
To differentiate whether an HCO
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Kinetics of n-[14C]butyrate uptake.
To further differentiate the mechanisms contributing to
HCO1 · 5 s
1. These data again
support the presence of an anion exchanger in the human colonic
basolateral membranes.
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Effect of transport inhibitors on n-[14C]butyrate
uptake.
To further assess the properties of n-[14C]butyrate
transport process across the colonic BLMV, effects of various membrane
ion transport inhibitors on the initial uptake rates of
n-[14C]butyrate were determined. Of the known membrane
ion transport inhibitors, the stilbene derivatives DIDS and SITS have
been shown to be the potent inhibitors of many anion exchange
processes. However, as shown in Fig. 3,
incubation of the BLMV with DIDS and SITS (5 mM) had no inhibitory
effect on HCO 0.05). Although, amiloride
(Na+/H+ exchange inhibitor) showed slight
inhibition of n-[14C]butyrate transport, acetazolamide
(carbonic anhydrase inhibitor) failed to alter the butyrate uptake in
the presence of an outwardly directed bicarbonate gradient. These
observations clearly indicate involvement of a distinct transporter in
the uptake of n-butyrate across the BLMV.
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Effect of anions on bicarbonate-stimulated
n-[14C]butyrate uptake.
To determine the anion specificity of the
HCO 0.05) of
HCO
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Transstimulation of n-[14C]butyrate uptake in human
colonic BLMV.
To further determine whether a carrier-mediated anion exchange process
accounts for HCO
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Effect of intravesicular and extravesicular Cl on
n-[14C]butyrate uptake.
Previous membrane vesicle studies have identified distinct
electroneutral Cl
/HCO
-independent
pathway for HCO
in the
intravesicular and extravesicular reaction media on butyrate transport
were also determined. As shown in Fig. 6,
in these experiments, the uptake of n-butyrate into the vesicles in the
presence of an outward- as well as inward-directed chloride gradient
was similar to that observed in the absence of a Cl
gradient (P > 0.05). Lack of inhibition of
HCO
uptake across the colonic
basolateral epithelia.
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DISCUSSION |
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SCFAs (acetate, propionate, and butyrate) are mainly produced as a result of microbial fermentation of dietary fiber in the lumen of the mammalian colon (4, 8-10). Although SCFAs have been shown to play a vital role in maintaining colonic health and in preventing colonic diseases (3, 13, 17, 27, 28), a clear understanding of the mechanisms of SCFA absorption across the colonic epithelial membrane domains and their further metabolism inside the colonocytes has not yet been well established. A number of previous in vivo (18, 24) and in vitro studies (5, 15, 19, 22, 38, 43) have suggested that the SCFA absorption across the intestinal apical domain involves either the nonionic diffusion of protonated SCFAs and/or a carrier-mediated anion exchange system. However, the contribution of the simple diffusion (5) versus anion exchange pathway (15, 16) under in vivo conditions is not entirely clear. Additionally, the mechanism of transport of the unmetabolized SCFA, across the human colonic basolateral domains is not well understood. Studies have shown extensive metabolic use of SCFAs by liver (26) and other peripheral tissues, thereby suggesting a probable existence of a transport mechanism for SCFA movement across the basolateral membrane of the colonocytes.
Recent studies from our laboratory and others utilizing vesicle
techniques have demonstrated the existence of a distinct
carrier-mediated HCO
Further evidence supporting the involvement of a distinct carrier in
HCO1 · 5 s
1. Although, the Km value
(17.5 ± 4.5 mM) for butyrate in the human colonic BLMVs is
comparable to the Km (26.9 mM) of butyrate for the previously characterized exchanger in rat BLMVs (30),
it appears to be significantly higher than Km
for butyrate exchanger in AMVs in both the human (1.5 ± 0.2 mM)
(15) and rat colon (6.9 mM) (22). Also, the
present data of the anion-specificity of the
HCO
Recent membrane vesicle studies from our laboratory have identified
distinct electroneutral Cl/HCO
uptake. In the
presence of high concentrations of extravesicular as well as
intravesicular Cl
no inhibition was observed in the
butyrate uptake into the BLMV compared with the uptake in the absence
of a Cl
gradient. However, these results are not in
agreement with the observations of Rajendran and Binder
(29), who demonstrated a Cl
-dependent SCFA
uptake system in rat colonic AMVs. The observed discrepancy in
n-butyrate uptake across the colonic epithelia in relation to the
effect of chloride may be due to species differences. Species
differences with respect to chloride dependence as well as DIDS
sensitivity (described above) could also be due to diverse dietary
habits in different species. In this regard, differences in transport
patterns of electrolytes in various regions of the intestine in
different species have also been observed (12, 20, 29,
36).
The HCO
In summary, our results provide a clear evidence for the existence of a distinct SCFA/anion exchanger on the basolateral membranes of the human proximal colonic epithelial cells, which appears to be distinct and separate from other previously characterized anion exchangers in the membranes of colonic epithelia. Further molecular identification and characterization of the anion-butyrate carrier in the human colonic basolateral membranes will be of great significance for understanding its role in SCFA absorption across the colonic epithelia and their relationship to the other colonic transporters.
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ACKNOWLEDGEMENTS |
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These studies were supported by the Department of Veterans Affairs and the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33349 (to K. Ramaswamy) and DK-54016 (to P. K. Dudeja).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. K. Dudeja, Associate Professor of Physiology in Medicine, University of Illinois at Chicago, Medical Research Service (600/151), Veterans Administration Medical Center, 820 S. Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.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.
10.1152/ajpgi.00173.2000
Received 26 April 2000; accepted in final form 18 December 2000.
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