Apical Membrane Of Caco-2 Cells Research Articles

Inhibitory Effect of 5,7-Dimethoxyfl avone On Rosuvastatin Uptake From The Apical Membrane Of Caco-2 Cells

5,7-Dimethoxyfl avone (5,7-DMF) is a natural polymethoxyfl avone, and acts as an inhibitor of ABC efflux transporters (BCRP, MRP2 and/ or P-gp), and rosuvastatin is taken up via OATP2B1 and secreted by BCRP and MRP2. In this study, we investigated the effect of 5,7-DMF on the transport of rosuvastatin through the apical membrane and the accumulation in Caco-2 cells. Furthermore, we investigated whether the rosuvastatin accumulation is mediated by monocarboxylate transporter 1 (MCT1), in addition to OATP2B1. Coincubation with 5,7- DMF significantly increased the cellular accumulation of rosuvastatin from the apical membranes of Caco-2 cells cultured on the plastic dish. Coincubation with Ko-143 (a BCRP inhibitor) or MK-571 (an MRP inhibitor) significantly increased the rosuvastatin accumulation, whereas coincubation with verapamil (a P-gp inhibitor) did not. Coincubation with benzoic acid or pravastatin, which are substrates of both OATP2B1 and MCT1, signifi cantly decreased the rosuvastatin accumulation, whereas coincubation with estron-3-sulfate or sulfobromophthalein, which are substrates of both OATP2B1 and ABC efflux transporter, did not decrease and increased the rosuvastatin accumulation, respectively. On the other hand, the transcellular transport of rosuvastatin from basolateral to apical side (B-to-A transport) was markedly higher than that from apical to basolateral side (A-to-B transport). Coincubation with 5,7-DMF from the apical side significantly increased the A-to-B transport and the accumulation of rosuvastatin, whereas that from the basolateral side significantly decreased the B-to-A transport of rosuvastatin with increases in the accumulation. These results suggest that 5,7-DMF may increase the rosuvastatin accumulation as a result of the inhibition of rosuvastatin efflux mediated by BCRP and MRP2, and the rosuvastatin transport through the apical membrane may be mediated by not only OATB2B1 but also MCT1.

NPC1L1-dependent transport of 27-alkyne cholesterol in intestinal epithelial cells.

Niemann-Pick C1 Like-1 (NPC1L1) mediates the uptake of micellar cholesterol by intestinal epithelial cells and is the molecular target of the cholesterol-lowering drug ezetimibe (EZE). The detailed mechanisms responsible for intracellular shuttling of micellar cholesterol are not fully understood due to the lack of a suitable NPC1L1 substrate that can be traced by fluorescence imaging and biochemical methods. 27-Alkyne cholesterol has been previously shown to serve as a substrate for different cellular processes similar to native cholesterol. However, it is not known whether alkyne cholesterol is absorbed via an NPC1L1-dependent pathway. We aimed to determine whether alkyne cholesterol is a substrate for NPC1L1 in intestinal cells. Human intestinal epithelial Caco2 cells were incubated with micelles containing alkyne cholesterol in the presence or absence of EZE. Small intestinal closed loops in C57BL/6J mice were injected with micelles containing alkyne cholesterol with or without EZE. Alkyne cholesterol esterification in Caco2 cells was significantly inhibited by EZE and by inhibitor of clathrin-mediated endocytosis Pitstop 2. The esterification was similarly reduced by inhibitors of the acyl-CoA cholesterol acyltransferase (ACAT). Alkyne cholesterol efficiently labeled the apical membrane of Caco2 cells and the amount retained on the membrane was significantly increased by EZE as judged by accessibility to exogenous cholesterol oxidase. In mouse small intestine, the presence of EZE reduced total alkyne cholesterol uptake by ∼75%. These data show that alkyne cholesterol acts as a substrate for NPC1L1 and may serve as a nonradioactive tracer to measure cholesterol absorption in both in vitro and in vivo models.

Effects of perfluoroalkyl carboxylic acids on the uptake of sulfobromophthalein via organic anion transporting polypeptides in human intestinal Caco-2 cells

We performed a detailed investigation of the uptake of sulfobromophthalein (BSP) from the apical membrane of Caco-2 cells, which is a substrate for organic anion transporting polypeptides (OATPs), and calculated the kinetic parameters of BSP uptake as follows: Km = 13.9 ± 1.3 μM, Vmax = 1.15 ± 0.07 nmol (mg protein)−1 (5 min)−1, and kd = 38.2 ± 0.53 μL (mg protein)−1 (5 min)−1. Coincubation with medium-chain (C7–C11) perfluoroalkyl carboxylic acids (PFCAs), such as perfluoroheptanoic acid (PFHpA, C7), perfluorooctanoic acid (PFOA, C8), perfluorononanoic acid (PFNA, C9), perfluorodecanoic acid (PFDA, C10) and perfluoroundecanoic acid (PFUnDA, C11), significantly decreased BSP uptake by 27–55%, while coincubation with short- (C3–C6) and long-chain (C12–C14) PFCAs decreased the uptake only slightly. Dixon plotting suggested that PFOA, PFNA and PFDA competitively inhibited the BSP uptake with inhibition constant (Ki) values of 62.2 ± 1.3 μM, 35.3 ± 0.1 μM and 43.2 ± 0.3 μM, respectively. PFCAs with medium-chains could be substrates for OATPs, probably OATP2B1, which is the most abundantly expressed OATP isoform in Caco-2 cells.

DHA and its derived lipid mediators MaR1, RvD1 and RvD2 block TNF-α inhibition of intestinal sugar and glutamine uptake in Caco-2 cells

Tumor necrosis factor-alfa (TNF-α) is a pro-inflammatory cytokine highly-involved in intestinal inflammation. Omega-3 polyunsaturated fatty acids (n3-PUFAs) show anti-inflammatory actions. We previously demonstrated that the n3-PUFA EPA prevents TNF-α inhibition of sugar uptake in Caco-2 cells. Here, we investigated whether the n3-PUFA DHA and its derived specialized pro-resolving lipid mediators (SPMs) MaR1, RvD1 and RvD2, could block TNF-α inhibition of intestinal sugar and glutamine uptake. DHA blocked TNF-α-induced inhibition of α-methyl-D-glucose (αMG) uptake and SGLT1 expression in the apical membrane of Caco-2 cells, through a pathway independent of GPR120. SPMs showed the same preventive effect but acting at concentrations 1000 times lower. In diet-induced obese (DIO) mice, oral gavage of MaR1 reversed the up-regulation of pro-inflammatory cytokines found in intestinal mucosa of these mice. However, MaR1 treatment was not able to counteract the reduced intestinal transport of αMG and SGLT1 expression in the DIO mice. In Caco-2 cells, TNF-α also inhibited glutamine uptake being this inhibition prevented by EPA, DHA and the DHA-derived SPMs. Interestingly, TNF-α increased the expression in the apical membrane of the glutamine transporter B0AT1. This increase was partially blocked by the n-3 PUFAs. These data reveal DHA and its SPMs as promising biomolecules to restore intestinal nutrients transport during intestinal inflammation.

Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides

The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na+ independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated Km and uptake clearance (Vmax/Km) values for PFOA were 8.3μM and 55.0μL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (Kd: 18.1μL/mg protein/min). Lineweaver–Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a Ki value of 23.1μM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP.

Effect of quercetin on the uptake and efflux of aristolochic acid I from Caco-2 cell monolayers.

The purpose of this study was to determine whether quercetin decreases the uptake of aristolochic acid I (AAI) from the apical membranes of Caco-2 cells via H(+) -linked MCTs at neutral pH as well as to confirm the secretion of AAI through the Caco-2 cell monolayers via ABC transporters. Caco-2 cells cultured on the dishes or permeable membranes were incubated with AAI in the absence or presence of quercetin or transporter inhibitors. Coincubation with quercetin decreased the uptake of AAI by Caco-2 cells cultured on the dishes at pH 7.4, and a similar decrease in AAI uptake was found when the cells were coincubated with acetic acid or benzoic acid. In contrast, the basolateral-to-apical transport of AAI was higher than the apical-to-basolateral transport of AAI at pH 7.4, and the former transport was decreased by quercetin and the BCRP inhibitors of Ko-143 and mitoxantrone, but not by P-gp or MRP2 inhibitors. AAI appears to be secreted from the apical membranes of Caco-2 cells via BCRP at neutral pH, although a small amount of AAI is taken up from the apical membranes via H(+) -linked MCTs, and quercetin may decrease both the BCRP-mediated efflux and the MCT-mediated influx of AAI.

Steric hindrance of 2,6-disubstituted benzoic acid derivatives on the uptake via monocarboxylic acid transporters from the apical membranes of Caco-2 cells

Benzoic acid is a typical substrate for monocarboxylic acid transporters (MCTs), and easily taken up from the apical membranes of Caco-2 cells by MCTs. However, some benzoic acid derivatives were sparingly taken up by Caco-2 cells. To elucidate the mechanism of lower uptake of the derivatives, we investigated the effect of substitution of benzene ring on the uptake by MCTs using Caco-2 cells. Among the benzoic acid derivatives tested, the uptake of 2,6-disubstituted benzoic acids was markedly lower than that of other benzoic acids. Co-incubation of the 2,6-disubstituted derivatives with benzoic acid did not decrease the uptake of benzoic acid, while co-incubation with other derivatives significantly decreased the uptake of benzoic acid. Kinetic analyses elucidated that the uptake of 2,6-dichlorobenzoic acid and 2,3,6-trichlorobenzoic acid did not involve the carrier-mediated process. The 2,6-disubstitution of benzoic acid may prevent the access of carboxylic acid group to MCTs expressed on the apical membranes of Caco-2 cells.

Uptake of aristolochic acid I into Caco-2 cells by monocarboxylic acid transporters.

The uptake mechanism of aristolochic acid I (AAI) was investigated using Caco-2 cells cultured on dishes and permeable membranes. The uptake of AAI from the apical membrane of Caco-2 cells cultured on a dish was rapid, and a decrease in the pH of the incubation medium significantly increased uptake. Incubation at low temperature (4°C) and treatment with sodium azide (a metabolic inhibitor) or carbonylcyanide p-trifluoromethoxyphenylhydrazone (a protonophore) significantly inhibited the AAI uptake. Coincubation with L-lactic acid or benzoic acid, typical substrates for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the AAI uptake, as did coincubation with α-cyano-4-hydroxycinnamate (an inhibitor of MCTs). Dixon plotting revealed the competitive inhibition of benzoic acid on the AAI uptake. To confirm the AAI uptake via MCTs, the apical-to-basolateral transport of AAI was investigated using the Caco-2 cells cultured on the permeable membranes. The transport of AAI at pH 6.0 was markedly higher than that at pH 7.4, and was significantly decreased by coincubation with benzoic acid. These results suggest that the uptake of AAI from the apical membrane of Caco-2 cells is mediated mainly by MCTs along with benzoic acid.

Carrier-mediated uptake of nobiletin, a citrus polymethoxyflavonoid, in human intestinal Caco-2 cells

The mechanism of intestinal absorption of nobiletin (NBL) was investigated using Caco-2 cells. The uptake of NBL from the apical membranes of Caco-2 cells was rapid and temperature-dependent and the presence of metabolic inhibitors, NaN3 and carbonylcyanide p-trifluoromethoxyphenylhydrazone, did not cause a decrease in NBL uptake. The relationship between the initial uptake of NBL and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The Km and uptake clearance (Vmax/Km) values for NBL were 50.6 and 168.1μl/mg protein/min, respectively. This clearance value was about 9-fold greater than that of the non-saturable uptake clearance (Kd: 18.5μl/mg protein/min). The presence of structurally similar compounds, such as quercetin and luteolin, competitively inhibited NBL uptake. These results suggest that uptake of NBL from the apical membranes of Caco-2 cells is mainly mediated by an energy-independent facilitated diffusion process.

Extracellular metabolism-dependent uptake of lysolipids through cultured monolayer of differentiated Caco-2 cells

Glycerophospholipids are known to be hydrolyzed in the intestinal lumen into free fatty acids and lysophospholipids that are then absorbed by the intestinal epithelial cells. A monolayer of enterocyte-differentiated Caco-2 cell is often used to assess the intestinal bioavailability of nutrients. In this study, we examined how differentiated Caco-2 cells process lysoglycerolipids such as lysophosphatidylcholine (LPC). Our findings were twofold. (1) Caco-2 cells secreted both a lysophospholipase A-like enzyme and a glycerophosphocholine-phosphodiesterase enzyme into the apical, but not basolateral, lumen, suggesting that food-derived LPC is converted to a free fatty acid, sn-glycerol-3-phosphate, and choline through two sequential enzymatic reactions in humans. The release of the latter enzyme was differentiation-dependent. (2) Fatty acid-releasing activities toward exogenous fluorescent LPC, lysophosphatidic acid and monoacylglycerol were shown to be higher on the apical membranes of Caco-2 cells than on the basolateral membranes. These results suggest that human intestinal epithelial cells metabolize lysoglycerolipids by two distinct mechanisms involving secreted or apical-selective expression of metabolic enzymes.

Absorption and Metabolism Characteristics of Rutin in Caco-2 Cells

The intestinal absorption and metabolism characteristics of the potentially beneficial polyphenol rutin were studied by measuring the intracellular accumulation and transport of rutin into Caco-2 cells with the sensitive and reliable analytical method of HPLC-coupled tandem mass spectrometry. Rutin and glucuronidated rutin were absorbed differently by the basolateral and apical membranes, and rutin showed differential permeability through the apical and basolateral sides. Approximately 33% of the rutin was metabolized to glucuronidated rutin, and the intracellular concentration of glucuronidated rutin was much lower than that of parent rutin. P-glycoprotein and multidrug-resistant proteins 2 and 3 were involved in the transmembrane transport and intracellular accumulation of rutin by Caco-2 cells. These results suggest that a specific transport system mediates rutin movement across the apical membrane in Caco-2 cells and that metabolic enzymes are important for this process.

Uptake of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) from the apical membranes of the human intestinal Caco-2 cells

We investigated whether the uptake of triclopyr (3, 5, 6-trichloro-2-pyridinyloxyacetic acid) and dicamba (3,6-dichloro-2-methoxybenzoic acid) across the apical membrane of Caco-2 cells was mediated via proton-linked monocarboxylic acid transporters (MCTs). The uptake of triclopyr from the apical membranes was fast, pH-, temperature-, and concentration dependent, required metabolic energy to proceed, and was competitively inhibited by monocarboxylic acids such as benzoic acid and ferulic acid (substrates of L-lactic acid-insensitive MCTs), but not by L-lactic acid. Thus, the uptake of triclopyr in Caco-2 cells appears to be mediated mainly via L-lactic acid-insensitive MCTs. In contrast, the uptake of dicamba (a benzoic acid derivative) was slow, and it was both pH- and temperature dependent. Coincubation with ferulic acid did not decrease the uptake of dicamba, although coincubation with benzoic acid moderately decreased it. The uptake of dicamba appears to be mediated mainly via passive diffusion, which is in contrast to the uptake of benzoic acid via MCTs. We speculate that the substituted groups in dicamba may inhibit uptake via MCTs.

Mechanisms for membrane transport of metformin in human intestinal epithelial Caco-2 cells

The aim of the present study was to investigate the mechanisms for membrane transport of metformin in human intestinal epithelial Caco-2 cells. The mRNA of not only organic cation transporter (OCT) 3, but also OCT1 and OCT2, was expressed in Caco-2 cells. The uptake of 100 µm metformin at the apical membrane of Caco-2 cells grown on porous filter membrane was significantly greater than that at the basolateral membrane. The apical uptake of 100 µm metformin in Caco-2 cells grown on plastic dishes was inhibited significantly by 1 mm unlabeled metformin, quinidine and pyrilamine, indicating that a specific transport system is involved in the apical uptake of metformin in Caco-2 cells. The apical uptake of 100 µm metformin in Caco-2 cells was decreased by acidification of the medium, but not increased by alkalization. In addition, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (a protonophore) had no effect on the apical uptake of metformin in Caco-2 cells at apical medium pH 8.4. These findings suggested that the apical uptake of metformin in Caco-2 cells is mediated at least partly by OCTs, but that the postulated H(+) /tertiary amine antiport system is not responsible for the apical uptake of metformin.

Role of monocarboxylic acid transporters in the cellular uptake of NSAIDs

The present study investigated the cellular uptake mechanism of non-steroidal anti-inflammatory drugs (NSAIDs) in Caco-2 cells. Diflunisal, diclofenac, ketoprofen and naproxen exhibited a strong inhibition effect on the cellular uptake of [14C]-benzoic acid in Caco-2 cells with IC50 values of 0.05-0.44 mM. The inhibition of naproxen and ketoprofen against the membrane transport of [14C]-benzoic acid appeared to be competitive, with Ki values of 0.22 and 0.38 mM, respectively. The membrane permeability of naproxen and ketoprofen was concentration dependent, implying that the cellular uptake pathway of ketoprofen and naproxen was saturable at the higher concentration. Furthermore, the cellular accumulation of ketoprofen was significantly reduced in the presence of benzoic acid and L-lactic acid, two known substrates of monocarboxylic acid transporter 1 (MCT1). These results suggest that MCT1 contributes at least in part to the carrier-mediated transport of NSAIDs containing a carboxylic acid moiety across the apical membrane in Caco-2 cells.

A flippase-independent function of ATP8B1, the protein affected in familial intrahepatic cholestasis type 1, is required for apical protein expression and microvillus formation in polarized epithelial cells

Mutations in ATP8B1 cause familial intrahepatic cholestasis type 1, a spectrum of disorders characterized by intrahepatic cholestasis, reduced growth, deafness, and diarrhea. ATP8B1 belongs to the P(4) P-type adenosine triphosphatase (ATPase) family of putative aminophospholipid translocases, and loss of aminophospholipid asymmetry in the canalicular membranes of ATP8B1-deficient liver cells has been proposed as the primary cause of impaired bile salt excretion. To explore the origin of the hepatic and extrahepatic symptoms associated with ATP8B1 deficiency, we investigated the impact of ATP8B1 depletion on the domain-specific aminophospholipid translocase activities and polarized organization of polarized epithelial Caco-2 cells. Caco-2 cells were stably transfected with short hairpin RNA constructs to block ATP8B1 expression. Aminophospholipid translocase activity was assessed using spin-labeled phospholipids. The polarized organization of these cells was determined by pulse-chase analysis, cell-fractionation, immunocytochemistry, and transmission electron microscopy. ATP8B1 was abundantly expressed in the apical membrane of Caco-2 cells, and its expression was markedly induced during differentiation and polarization. Blocking ATP8B1 expression by RNA interference (RNAi) affected neither aminophospholipid transport nor the asymmetrical distribution of aminophospholipids across the apical bilayer. Nonetheless, ATP8B1-depleted Caco-2 cells displayed profound perturbations in apical membrane organization, including a disorganized apical actin cytoskeleton, a loss in microvilli, and a posttranscriptional defect in apical protein expression. Our findings point to a critical role of ATP8B1 in apical membrane organization that is unrelated to its presumed aminophospholipid translocase activity, yet potentially relevant for the development of cholestasis and the manifestation of extrahepatic features associated with ATP8B1 deficiency.

Mechanisms for Transport of Methotrexate across Apical and Basolateral Membranes in Human Intestinal Epithelial Caco-2 Cells

To investigate the intestinal absorption mechanisms of methotrexate,we examined the apical uptake and transcellular transport of the drug in human intestinal epithelial Caco-2 cells.In these cells,the mRNA expression of proton-coupled folate transporter (PCFT) was much higher than that of reduced folate carrier (RFC).The apical uptake of 1μM methotrexate by Caco-2 cells grown on plastic dishes was markedly increased by acidification (pH 6.5→4.citation=5)of the apical medium,and diminished at a low temperature (4°C).The transcellular transport of 1μM methotrexate across Caco-2 cell monolayers grown on porous membrane filters was only slightly greater in the apical-to-basolateral direction than in the opposite direction.Pharmacokinetic analysis of the transcellular transport revealed that the influx clearance of methotrexate at the apical membrane was greater than that at the basolateral membrane,with significant gradual decreases in the low concentration range (1→10μM).On the other hand,the efflux clearance of methotrexate was much lower than the influx clearance at both apical and basolateral membranes.These findings indicated that high-affinity PCFT was involved in the uptake of methotrexate across the apical membrane in Caco-2 cells,and that there was little or no efflux of the drug at the basolateral membrane.

Intestinal Absorption and Secretion Mechanism of Carboxylate Drugs

Oral drug delivery is generally the most desirable means of administration, mainly because of patient acceptance, convenience in administration. Intestinal absorption mechanisms of anionic drugs have been mainly explained by the passive diffusion of nonionized compounds. However, several studies have suggested the involvement of specific transporters in intestinal absorption of weak acids including monocarboxylates. (-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a oral hypoglycemic agent possessing a carboxyl group and a peptide-type bond in its structure. Although nateglinide quickly reaches the maximal serum concentration after oral administration, nateglinide itself is not transported by PepT1 or MCT1. We demonstrated that nateglinide transport occurs via a single system that is H(+) dependent but is distinct from PepT1 or MCT1. In clinical, patients usually take many kinds of drugs at the same time. Thus, drug-drug interactions involving transporters can often directly affect the therapeutic safety and efficacy of many drugs. However, there have been few studies on food-drug interactions involving transporters. Dietary polyphenols have been widely assumed to be beneficial to human health. Polyphenols are commercially prepared and used as functional foods. We reported that ferulic acid, which is widely used as a functional food, affects the transport of clinical agents. The major dose-limiting toxicity after administration of irinotecan hydrochloride, 7-ethyl-10-(4-[1-piperidino]-1-piperidino)-carbonyloxycamptothecin (CPT-11) is severe diarrhea. We have found that a specific transport system mediates the uptake of active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) across the apical membrane in Caco-2 cells. Baicalin and sulfobromophthatlein inhibit this transporter. Inhibition of this transporter would be a useful means for reducing late-onset diarrhea.

Uptake of phenoxyacetic acid derivatives into Caco-2 cells by the monocarboxylic acid transporters

The uptake mechanism of phenoxyacetic acid (PA) and its chlorine derivatives, 4-chlorophenoxyacetic acid (4-CPA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was investigated using Caco-2 cells. The cells were incubated with PA, 4-CPA, 2,4-D or 2,4,5-T at pH 6.0 and 37 degrees C. The order of uptake and lipophilicity expressed by n-octanol partition coefficients were PA<4-CPA<2,4-D<2,4,5-T. Incubation at 4 degrees C or at pH 7.4 significantly decreased these uptake. Furthermore, pretreatment with the protonophore, carbonylcyanide-p-(trifluoromethoxy) phenylhydrazone, or coincubation with benzoic acid, a typical substrate for the proton-linked monocarboxylic acid transporters (MCTs), significantly decreased the uptake of all compounds. The initial uptake rates of all compounds except PA were apparently saturable, suggesting the involvement of a carrier-mediated process. The order of uptake clearance of the compounds was the same as the order of their uptake and lipophilicity. Preloading of cells with benzoic acid significantly increased their uptake except for PA. These results suggest that the uptake of PA, 4-CPA, 2,4-D and 2,4,5-T from the apical membrane of Caco-2 cells is mediated via common MCTs shared, at least in part, with benzoic acid, and the increase in lipophilicity due to the chlor-substitution may increase uptake via the MCTs.

W1742 Mechanisms of Human Hepatic Vitamin C Uptake: Promoter Analysis and Adaptive Regulation of the Hsvct Systems

carrier system in apical membrane. Phloretin, a GLUT2 inhibitor, significantly inhibited glucose uptake, especially at high glucose concentration and in incubation durations of >1 min raising the possibility that the increased Km and Vmax were due to translocation of GLUT2. Western blotting of the apical membrane using antibodies against SGLT-1 and GLUT2 suggested that GLUT2 rather than SGLT-1 translocated to apical membrane. Nocodazole inhibited carrier-mediated glucose uptake at high glucose concentrations (25 and 50 mM) during the 5 and 10 min incubation, but not at low glucose concentrations (0.5-10 mM) or with short durations of exposure (30 s and 1 min). CONCLUSION: GLUT2 is translocated to the intestinal apical membrane of Caco-2 cells via a glucose-dependent mechanism acting via the microtubular system. (Support: NIH DK39337-MGS)

Taurine

Taurine, a sulfur-containing amino acid (Figure), has been termed a functional nutrient that could be used to protect against, among others, diabetes mellitus and atherosclerosis.1 Indeed, an increasing body of literature supports the use of taurine supplements. Because taurine has very diverse functions, notably, intracellular osmoregulation and bile acid formation, and is abundantly present in several organs, multiple pathways could be involved. Some of these are discussed in this editorial. Figure. A simplified biosynthesis pathway from methionine to taurine. Methionine is converted to homocysteine in 3 steps. Homocysteine is used as a substrate to form cysteine in 2 steps but can also be converted back to methionine by 2 different mechanisms. Cysteine, precursor for the important antioxidant glutathione and the gaseous transmitter/signaling molecule H2S, can be further converted to cysteine sulfinate. Cysteine sulfinate is converted to taurine in 2 steps via 2 different pathways. Taurine can decrease methionine by decreasing uptake. Whether taurine decreases homocysteine directly or via a reduction of methionine is unclear. Of the 20 canonical amino acids, 18 are composed of carbon, hydrogen, nitrogen, and oxygen only. The remaining 2, methionine and cysteine, also contain 1 atom of sulfur. Because sulfur is not as electronegative as oxygen, the sulfur-containing amino acids play a key role in protein structure and synthesis.2 Methionine and cysteine also play important roles in cell metabolism. For instance, methionine serves as a substrate for S-adenosylmethionine, which is vital for methylation of nucleic acids, proteins, lipids, etc. In proteins, cysteine easily forms double bonds with other cysteine residues, thus determining tertiary structure and binding sites. Moreover, cysteine is substrate for glutathione, an important intracellular antioxidant, and H2S, a gas that can induce endothelial-dependent relaxation.3 Cysteine is considered a nonessential amino acid because it is synthesized …