Effect of ethanol on intestinal calcium transport in chicks

To test the hypothesis that chronic ethanol exposure would alter the membrane fluidity of the intestinal brush-border membrane, which would lead to calcium malabsorption, we gave chicks 15% ethanol in their drinking water from hatching to 3 or 4 wk of age. Although such chicks grew less quickly than their hatchmates not ingesting ethanol, their ability to absorb calcium from the duodenum in vivo was unimpaired. However, when calcium accumulation by isolated duodenal brush-border membrane vesicles (BBMVs) was assessed, the BBMVs from chicks ingesting ethanol for 1-3 wk had a lower rate of uptake than the BBMVs from the controls at all calcium concentrations evaluated (0.1-25 mM). This difference could not be explained by differences in membrane fluidity as assessed by fluorescence depolarization, or by changes in intravesicular volume. Glucose uptake was not affected. The acute addition of ethanol (up to 1 M) in vitro to the control BBMVs increased both membrane fluidity and calcium accumulation. No difference in the fluidizing effect of ethanol in BBMVs between ethanol-ingesting chicks and control chicks could be demonstrated, although the acute effect of ethanol on calcium accumulation was blunted in the BBMVs from chicks ingesting ethanol. Increasing the temperature of the incubation medium also increased membrane fluidity and calcium accumulation in BBMVs from control and ethanol-ingesting chicks, with a greater increase in calcium uptake by the control BBMVs despite comparable increases in fluidity in BBMVs from the control and ethanol-ingesting chicks. We conclude that the chronic ingestion of ethanol by chicks, although markedly altering growth rates, has minimal impact on the intestinal absorption of calcium when assessed in vivo. However, chronic ethanol ingestion does appear to alter the intestinal brush-border membrane to make it less permeable to calcium and less susceptible to the stimulation by ethanol of calcium flux across this membrane; this adaptation may prevent increased flux of calcium across the brush-border membrane into the cell in the presence of ethanol.     

Independent modulation by food supply of two distinct sodium-activated D-glucose transport systems in the guinea pig jejunal brush-border membrane.

D-glucose transport across the intestinal brush-border membrane involves two transport systems designated here as systems 1 and 2. Kinetic properties for both D-glucose and methyl alpha-D-glucopyranoside transport were measured at 35 degrees C by using brush-border membrane vesicles prepared from either control, fasted (48 hr), or semistarved (10 days) animals. The results show the following: (i) The sugar influx rate by simple diffusion was identical under either altered condition. (ii) Semistarvation stimulated D-glucose uptake by system 2 (both its Vmax and Km increased), whereas system 1 was untouched. (iii) Fasting increased the capacity of system 1 without affecting either Km of system 1 or Vmax and Km of system 2. The effect of fasting on Vmax of system 1 cannot be attributed to indirect effects from changes in ionic permeability because the kinetic difference between control and fasted animals persisted when the membrane potential was short-circuited with equilibrated K+ and valinomycin. This work provides further evidence for the existence of two distinct sodium-activated D-glucose transport systems in the intestinal brush-border membrane, which adapt independently to either semistarvation or fasting.     

Hormonal regulation of dipeptide transporter （PepT1） in Caco-2 cells with normal and anoxia／reoxygenation management

AIM: To determine the regulation effects of recombinant human growth hormone (rhGH) on dipeptide transporter(PepT1) in Caco-2 cells with normal culture and anoxia/reoxygenation injury. METHODS: A human intestinal cell monolayer (Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter (PepT1). Caco-2 cells grown on Transwell membrane filters were preincubated in the presence of rhGH in the culture medium for 4 d, serum was withdrawn from monolayers for 24 h before each experiment. The transport experiments of cephalexin across apical membromes were then conducted; Caco-2 cells grown on multiple well dishes (24 pore) with normal culture or anoxia/reoxygenation injury were preincubated with rhGH as above and uptake of cephalexin was then measured. RESULTS: The transport and uptake of cephelaxin across apical membranes of Caco-2 cells after preincubation with rhGH were significantly increased compared with controls (P=0.045, 0.0223). Also, addition of rhGH at physiological concentration (34 nM) to incubation medium greatly stimulates cephalexin uptake by anoxia/reoxygenation injuried Caco-2 cells (P=0.0116), while the biological functions of PepT1 in injured Caco-2 cells without rhGH were markedly downregulated. Northern blot analysis showed that the level of PepT1 mRNA of rhGH-treated injured Caco-2 cells was greatly increased compared to controls. CONCLUSION: The present results of rhGH stimulating the uptake and transport of cephalexin indicated that rhGH greatly upregulates the physiological effects of dipeptide transporters of Caco-2 cells. The alteration in the gene expression may be a mechanism of regulation of PepT1. In addition, Caco-2 cells take up cephalexin by the Proton-dependent dipeptide transporters that closely resembles the transporters present in the intestine. Caco-2 cells represent an ideal cellular model for future studies of the dipeptide transporter.     

Characteristics and mechanism of glutamine-dipeptide absorption in human intestine.

Using in vivo and in vitro techniques, the mechanism by which intestinal mucosa obtains glutamine from luminal oligopeptides was investigated in humans. The rate of hydrolysis by mucosal brush border membrane was more than threefold greater for alanylglutamine than for glycylglutamine. Despite this difference, rates of dipeptide and amino acid disappearance during intestinal perfusion were greater from test solutions containing glycylglutamine than alanylglutamine. Furthermore, rates of intraluminal appearance of products of hydrolysis during the infusion of two dipeptides were similar and less than 5% of the disappearance rate of the parent dipeptide. In contrast to free glutamine, uptake of peptide-bound glutamine by brush border membrane vesicles was not inhibited by deletion of sodium or addition of free amino acids to the incubation medium but was inhibited by other oligopeptides and stimulated by a proton gradient. Inhibition constants for the saturable uptake of glycylglutamine and alanylglutamine by vesicles were not significantly different, suggesting similar affinities for the peptide transporter. It is concluded that in human intestine the predominant mechanism for assimilation of glutamine-dipeptides is absorption as intact dipeptide rather than hydrolysis.     

Effects of non-steroidal anti-inflammatory drugs on the methotrexate transport in rat small intestine

BACKGROUND: Methotrexate (MTX) is used in the treatment of rheumatic disease, sometimes along with non-steroidal anti-inflammatory drugs (NSAIDs), and is actively co-transported with H+ in the small intestine, mediated by a reduced folate carrier (RFC). The co-administration of NSAIDs with MTX might cause a decrease in MTX absorption through the small intestine, since some NSAIDs are uncouplers of oxidative phosphorylation. The present study investigates whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decrease the ATP content of small intestinal epithelial cells and affect MTX transport (the secondary active transport) in the small intestine. METHODS: MTX transport was examined in the presence and absence of the NSAIDs, using the everted intestine technique and brush border membrane vesicles (BBMVs) from the rat small intestine. The change in physical properties of the membrane was studied in BBMVs using the fluorescence techniques. RESULTS: MTX absorption in the small intestine with H+ gradient (mucosal side, pH 6.0; serosal side, pH 7.4) decreased in the presence of the NSAIDs, but absorption without H+ gradient (both sides, pH 7.4) was unaffected. The intestinal mucosal ATP content decreased in the presence of the NSAIDs. The uptake of MTX in BBMVs was unaffected by the NSAIDs. The activity of intestinal Na+-K+-ATPase was enhanced in the presence of the NSAIDs. The fluorescence measurements showed that membrane fluidity, membrane potential and membrane hydrophobicity of BBMVs were unaffected by the NSAIDs. CONCLUSIONS: NSAIDs decreased the H+/MTX absorption in the small intestine, but not the passive transport. The uncoupling effect of the NSAIDs decreased the ATP content in the small intestine, resulting in inhibition of the secondary active transport.     

Inhibition of iron transport across human intestinal epithelial cells by hepcidin

We investigated the effects of the iron regulatory peptide hepcidin on iron transport by the human intestinal epithelial Caco-2 cell line. Caco-2 cells were exposed to hepcidin for 24 hours prior to the measurement of both iron transport and transporter protein and mRNA expression. Incubation with hepcidin significantly decreased apical iron uptake by Caco-2 cells. This was accompanied by a decrease in both the protein and the mRNA expression of the iron-response element containing variant of the divalent metal transporter (DMT1[+IRE]). In contrast, iron efflux and iron-regulated gene1 (IREG1) expression were unaffected by hepcidin. Hepcidin interacts directly with a model intestinal epithelium. The primary effect of this regulatory peptide is to modulate the apical membrane uptake machinery, thereby controlling the amount of iron absorbed from the diet.     

D-glucose uptake is increased in jejunal brush-border membrane vesicles from hyperthyroid chicks

Jejunal brush-border membrane vesicles were harvested from 4-week old chicks whose thyroid status had been altered either by a daily injection of 20 micrograms T3 for 1 week or which through the preceding 4 weeks had received propylthiouracil and that had been repleted with either 20 or 80 micrograms T3 in divided doses within 48 h. T3 markedly stimulated D-glucose uptake in brush-border membrane vesicles in the presence of an outside/inside (100/0 mmol/l) Na+ gradient. T3 administration had no detectable influence on the Na+ permeability of the isolated vesicles. The effect of the thyroid hormone on Na+ gradient-driven D-glucose uptake was fully preserved at zero transmembrane potential difference. These findings exclude that T3 stimulates Na+-dependent D-glucose transport in the small intestine through changes of the electrochemical Na+ gradient or through alteration of the transmembrane potential difference. Tracer exchange experiments under equilibrium and voltage-clamp conditions revealed a significantly shorter half-time of D-glucose uptake in brush-border membrane vesicles from T3-treated chicks. Kinetic analysis showed that T3 administration significantly increases the apparent maximal velocity of D-glucose transport in brush-border membrane vesicles, whereas the apparent Km values were virtually unaltered. From these data we conclude that T3 increases the activity of Na+-dependent D-glucose carriers in the brush-border membrane. This is interpreted as consistent with a greater rate of D-glucose absorption from the intestinal lumen under conditions of hyperthyroidism.     

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H⁺ gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H⁺ binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H⁺-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.     

Ethanol-induced inhibition of glucose transport across the isolated brush-border membrane of hamster jejunum

Acute exposure of jejunal mucosa to ethanol has been reported to produce a depression of transmural glucose transport across this organin vitro andin vivo. In an attempt to understand the mechanism of action of ethanol on intestinal transport, in the present study we have investigated the effect of ethanol on glucose uptake by purified brush-border membrane vesicles of hamster jejunum. Ethanol, in concentrations found in man after moderate drinking (1–5% w/v), was found to depress glucose uptake by the brush-border membrane in a dose-dependent and time-dependent manner. Mannose was used to measure nonspecific uptake, and we found that the ethanol-induced depression of glucose uptake was not related to an alteration of the nonspecific uptake of this sugar. The inhibition of glucose uptake of the ethanol-treated membranes completely disappeared after repeated washing of the membranes with ethanol-free buffer. Accordingly, the ethanol-induced depression of glucose uptake was not the result of irreversible damage to membrane proteins but was related to a direct effect of ethanol on the brush-border membrane. On the basis of these findings, it is concluded that a direct interference with glucose translocation across the brush border plays an important role in the ethanol-induced depression of transmural jejunal glucose absorption.

     

Responsive transporter genes within the murine intestinal-pancreatic axis form a basis of zinc homeostasis

Zn homeostasis in animals is a consequence of avid uptake and retention, except during conditions of limited dietary availability, and/or factors such as parasites, which compete for this micronutrient or compromise retention by the host. Membrane proteins that facilitate Zn transport constitute the SLC30A (ZnT) and SLC39A (Zip) gene families. Because dietary recommendations are based on the balance between intestinal absorption and endogenous losses, we have studied Zn transporter expression of the murine intestinal-pancreatic axis to identify transporters that are likely to be involved in homeostatic control of Zn metabolism. Marked tissue specificity of expression was observed in Zn-depleted vs. Zn-adequate mice. As shown by quantitative PCR, Western blot analysis, and immunohistochemistry, intestinal Zip4 was markedly up-regulated in response to Zn-depletion conditions. The increased abundance of Zip4 is concentrated at the apical membrane of enterocytes. There are 16 ZnT and Zip transporters expressed in pancreas. Only two, ZnT1 and ZnT2 (both cellular Zn exporters), show a progressive down-regulation under Zn-depleted conditions. In Zn-adequate mice, ZnT1 is diffusely distributed in acinar cell cytoplasm and colocalizes with alpha-amylase but is not detected in pancreatic islets. In acinar cells during Zn depletion, ZnT1 is localized to the plasma membrane. Intestinal Zip4 up-regulation by Zn-depletion conditions is dampened in metallothionein knockout mice, suggesting that intracellular Zn pools influence these responses. The results show that Zn transporter expression in the intestinal-pancreatic axis is a component of the homeostatic regulation of this micronutrient.     

Enteral feeding of glycyl-glutamine dipeptide improves the structure and absorptive function of the small intestine after allogenetic liver transplantation in rats

BACKGROUND: Recipients of liver transplantation could have postoperative structural injury and declined absorptive function in the gastrointestinal tract. Glutamine (Gln) is a special nutrient of small intestinal mucosa and of various kinds of cells proliferating rapidly. But Gln could form a kind of poisonous pyroglutamic acid in water solution, which is the limitation of Gln in clinical practice. Glycyl-glutamine (Gly-Gln) is highly soluble and can be hydro-lyzed to release glutamine. This study was undertaken to observe the effect of Gly-Gln dipeptide by enteral feeding on the intestinal structure and absorptive function after allogenetic liver transplantation in rats. METHODS: Twelve male inbred Lewis rats were selected randomly as donors, and 24 male inbred BN rats as recipients of allogenetic liver transplantation. The recipients were also randomly divided into two groups; control group (ALA group, n = 12 ) and experimental group ( GLN group , n =12). In each group, 6 normal BN rats were sampled as the normal parameter on the 3rd preoperative day. The 6 recipients in the control group received alanine 0. 6 g/kg daily for 3 days before operation and 7 days after operation by gastric perfusion, and the 6 recipients in the experimental group were given Gly-Gln 0.6 g/kg daily the same way. The 12 BN recipients underwent 3-day fasting (free access to water with 0. 23% sodium chloride) and ortho-topic liver transplantation in aseptic conditions and were given subcutaneous injection of CsA 2 mg/kg daily after the operation. The 12 BN recipients were sampled on the 8th postoperative day. All of the 24 BN rats were subjected to examination of mucosal structure, activities of Na + -K + - ATP and disaccharidase, and D-xylose absorption test. RESULTS: The 12 BN recipients were alive after liver transplantation. On the 3rd preoperative day, mucosal structure , activities of Na + -K -ATP and disaccharidase and D-xylose absorption in the two groups were not significantly different. On the 8th postoperative day, the parameters of the two groups were markedly changed compared with those on the 3rd preoperative day. However, the parameters of GLN group were remarkably higher than those of ALA group. CONCLUSION: Enteral feeding of Gly-Gln could improve the structure and absorptive function of the small intestine after liver transplantation in rats.     

Calmodulin-mediated effects of loperamide on chloride transport by brush border membrane vesicles from human ileum

We investigated whether the synthetic opiate loperamide-HCl is able to regulate specific transport systems for sodium and chloride in brush border membrane vesicles (BBMVs) from human ileum and whether such activities are mediated by calcium/calmodulin. In BBMVs we studied Na+/H+ antiport, Cl+/OH- antiport, Na+/Cl- cotransport, and the Cl- conductive pathway. Brush border membrane vesicles were incubated with 10 microM loperamide over 4 h at 5 degrees C before the uptake experiments. In ileal BBMVs, loperamide stimulated intravesicular accumulation of Na+ in the presence of Cl- and vice versa. After 1 min of incubation, the stimulatory effect was 35% +/- 5% (p less than 0.005) of the control without loperamide. Loperamide also stimulated Cl-/OH- antiport by 30% +/- 5% (p less than 0.005) in BBMVs of ileum. In addition, we studied the role of Ca2+/calmodulin in the action of loperamide on chloride transport by human BBMVs. In loperamide-pretreated BBMVs, calmodulin activity was significantly decreased (12 +/- 2 vs. 38 +/- 4 pmol/mg protein). When loperamide-pretreated vesicles were incubated with 2 microM calcium (free concentration) plus 5 microM calmodulin for 1 h at 5 degrees C, complete inhibition of the stimulatory effect of loperamide on Cl-/OH- antiport and Na+/Cl- cotransport was observed. Increasing the Ca2+/calmodulin activity of loperamide-pretreated BBMVs with 2 microM calcium plus 5 microM calmodulin led to a significant inhibition of Cl-/OH- antiport and Na+/Cl- cotransport by 40% +/- 5% (p less than 0.005).     

The locally acting glucocorticosteroid budesonide enhances intestinal sugar uptake following intestinal resection in rats

BACKGROUND AND AIMS: Locally and systemically acting corticosteroids alter the morphology and transport function of the intestine. This study was undertaken to assess the effect of budesonide, prednisone, and dexamethasone on sugar uptake. METHODS: Adult male Sprague Dawley rats underwent transection or resection of 50% of the middle portion of the small intestine, and in vitro uptake of sugars was measured. RESULTS: The 50% enterectomy did not alter jejunal or ileal uptake of glucose or fructose. Prednisone had no effect on the uptake of glucose or fructose in resected animals. In contrast, in resected rats budesonide increased by over 120% the value of the jejunal maximal transport rate for the uptake of glucose, and increased by over 150% ileal uptake of fructose. Protein abundance and mRNA expression of the sodium dependent glucose transporter in brush border membrane (SGLT1), sodium independent fructose transporter in the brush border membrane (GLUT5), sodium independent glucose and fructose transporter in the basolateral and brush border membranes (GLUT2), and Na(+)/K(+) ATPase alpha1 and beta1 did not explain the enhancing effect of budesonide on glucose or fructose uptake. Budesonide, prednisone, and dexamethasone reduced jejunal expression of the early response gene c-jun. In resected animals, expression of the mRNA of ornithine decarboxylase (ODC) in the jejunum was reduced, and corticosteroids reduced jejunal expression of the mRNA of proglucagon. CONCLUSIONS: These data suggest that the influence of corticosteroids on sugar uptake in resected animals may be achieved by post translational processes involving signalling with c-jun, ODC, and proglucagon, or other as yet unknown signals. It remains to be determined whether budesonide may be useful to stimulate the absorption of sugars following intestinal resection in humans.     

The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostalpha-Ostbeta exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostalpha-Ostbeta have not been investigated. To determine the role of Ostalpha-Ostbeta in intestinal bile acid absorption, the Ostalpha gene was disrupted by homologous recombination in mice. Ostalpha(-/-) mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostalpha(-/-) vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostalpha(-/-)Mrp3(-/-) mice. The bile acid pool size was significantly reduced (>65%) in Ostalpha(-/-) mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostalpha(-/-) mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostalpha-Ostbeta is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.     

Changes of biological functions of dipeptide transporter （PepT1） and hormonal regulation in severe scald rats

AIM: To determine the regulatory effects of recombinant human growth hormone (rhGH) on dipeptide transport (PepT1) in normal and severe scald rats. METHODS: Male Sprague-Dawley rats with 30 % total body surface area (TBSA) III degree scald were employed as the model. In this study rhGH was used at the dose of 2 IU.kg^1.d^-1 An everted sleeve of intestine 4 cm long obtained from mid-jejunum was securely incubated in Kreb‘s solution with radioactive dipeptide (3H-glycylsarcosine, 3H-Gly-Sar,10μCi/ml) at 37℃ for 15 min to measure the effects of uptake and transport of PepT1 of small intestinal epithelial cells in normal and severe scald rats. RESULTS: Abundant blood supply to intestine and mesentery was observed in normal and scald rats administered rhGH, while less supply of blood to intestine and mesentery was observed in rats without rhGH. Compared with controls, the transport of dipeptide in normal rats with injection of rhGH was not significantly increased (P=-0.1926), while the uptake was significantly increased (P=-0.0253). The effects of transport and uptake of PepT1 in scald rats with injection of rhGH were significantly increased (P=0.0082, 0.0391). CONCLUSION: Blood supply to intestine and mesentery of rats was increased following injection of rhGH. The effectsof uptake and transport of dipeptide transporters in small intestinal epithelial cells of rats with severe scald were markedly up-regulated by rhGH.     

Triiodothyronine stimulates 2-deoxy-D-glucose uptake by organ cultured embryonic chick small intestine

The possible contribution of increased D-glucose absorption from the intestine to the impairment of oral glucose tolerance in hyperthyroidism was evaluated by investigating the influence of T3 on different pathways of D-glucose transport, utilizing an organ culture system of embryonic chick small intestinal explants. T3, when present in the culture medium at a concentration between 10(-10)-10(-8) mol/l, had no effect on uptake of alpha-methyl-D-glucoside, but stimulated uptake of 2-deoxy-D-glucose by the intestinal epithelium in a dose-dependent fashion. T3 thereby enhanced the maximal velocity of a saturable, cytochalasin B-sensitive but phloretin-insensitive 2-deoxy-D-glucose transport system with an apparent Km of 7 mmol/l. The combined data are consistent with the assumption that T3 can enhance D-glucose entry into the intestinal epithelium through stimulation of a low-affinity transport system at the brush-border membrane of enterocytes. Our findings provide a basis for the explanation of adaptive modulation of intestinal glucose absorption in hyperthyroidism.     

Sulfate-sodium cotransport by brush-border membrane vesicles isolated from rat ileum

Uptake of inorganic sulfate into brush-border membrane vesicles isolated by a calcium precipitation method from rat small intestine was investigated using a rapid filtration technique and 35Sulfur acid as tracer. Sulfate uptake by membrane vesicles was osmotically sensitive, suggesting transport into an intravesicular space rather than binding to or incorporation into the membrane. Transport of sulfate into brush-border vesicles isolated from rat ileum was only stimulated by sodium ions as compared with other monovalent cations. A typical "overshoot" phenomenon was observed in the presence of an inwardly directed NaCl gradient. Tracer sulfate exchange was faster in the presence of sodium than in the presence of potassium. Addition of the ionophores for monovalent cations, monactin, or gramicidin D, decreased the sodium gradient-driven sulfate uptake. Sulfate uptake showed a saturation phenomenon only in the presence of sodium. Transstimulaton of sodium-dependent sulfate transport was shown with MoO4(2-), but not with PO4(2-) and WO4(2-). Changing the electrical potential difference across the membrane vesicles by establishing different diffusion potentials (anion replacement; potassium gradient +/- valinomycin) did not alter sodium-dependent sulfate uptake. Stimulation of sulfate transport by sodium was greater in membrane vesicles from ileal segments than from duodenum or jejunum. It is concluded that isolated brush-border membranes of rat ileum contain an electroneutrol sodium-sulfate cotransport system.     

Monocarboxylate 4 Mediated Butyrate Transport in a Rat Intestinal Epithelial Cell Line

Background

Short chain fatty acids (SCFA) are absorbed by carrier mediated uptake in the small intestine by pH-dependent SCFA/HCO₃ ^? exchangers on the apical membrane of epithelial cells. Conventional assumption is that MCT1 mediates SCFA/HCO₃ ^? exchange in the intestine. Further, due to the presence of multiple such anion exchangers, the identity of the intestinal SCFA/HCO₃ ^? has been controversial.

Aims

The aim of this study was to determine the identities of the butyrate transporter in the intestinal epithelial cells (IEC-18).

Methods

IEC-18 cells were treated with specific siRNAs for MCT1 and MCT4, and butyrate and lactate uptake studies were performed.

Results

Alpha-cyano-4-hydroxycinnamic acid inhibited lactate uptake but not butyrate uptake in IEC-18 cells, indicating that these two substrates are transported via two different transporter systems. MCT1 siRNA treatment abolished both MCT1 mRNA by more than 95 % and protein expression by 83 % as evidenced by RTQ-PCR and western blotting experiments. However, MCT1 siRNA treatment inhibited butyrate uptake upto 24 %, whereas it inhibited lactate uptake significantly by 70 %. Treatment with MCT4 siRNA inhibited MCT4 mRNA expression by 75 % and protein expression by 85 % in these cells. MCT4 siRNA inhibited butyrate uptake by 40 %. Further, several non-steroidal anti-inflammatory drugs (NSAIDs) are transported by the butyrate transporter. Finally, MCT4 siRNA inhibited salicylate uptake by 27 % indicating direct evidence for the transport of salicylate by MCT4.

Conclusions

These data indicate that MCT1 is the high affinity lactate transporter and MCT4 is the high affinity butyrate transporter in the intestinal epithelial cell line IEC-18.