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Purpose In Caco-2 cells, folate uptake via the proton-coupled folate transporter (PCFT) increases significantly by a 3-day treatment with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Additionally, mRNA content and protein expression of the transporter OATP1A2 were increased up to ninefold with 1,25(OH)2D3. We investigated whether these in vitro findings can be confirmed in humans in vivo. Methods Ten healthy volunteers (six women) received 5 mg folic acid orally once before and once together with the last intake of a 10-day course of 0.5 μg 1,25(OH)2D3 orally. One hundred twenty milligrams fexofenadine, an OATP1A2 substrate, was taken in 1 day before the first folic acid intake, and again on the ninth day of 1,25(OH)2D3 intake. Duodenal biopsies were taken for transporter mRNA assessments once before and once on the ninth or tenth day of the vitamin D3 course. Serum folic acid and fexofenadine concentrations were quantified with a chemiluminescence immunoassay and LC-MS/MS, respectively. Pharmacokinetics were compared between periods with standard bioequivalence approaches. Results While geometric mean folic acid AUC0-2h, which mainly reflects absorption, was 0.403 and 0.414 mg/L·h before and after the vitamin D3 course (geometric mean ratio (GMR), 1.027; 90 % confidence interval (90 % CI), 0.788–1.340), the geometric mean fexofenadine AUC0-2h was 1.932 and 2.761 mg/L·h, respectively (GMR, 1.429; 90 % CI, 0.890–2.294). PCFT- and OATP1A2-mRNA expressions in duodenal biopsies were essentially unchanged. Conclusions No significant changes in folic acid and fexofenadine absorption were observed after a 10-day course of 1,25(OH)2D3 in humans in vivo. This study underlines the importance of confirming in vitro findings in vivo in humans.

Intestinal absorption of drugs, nutrients, and other compounds is mediated by uptake transporters expressed at the apical enterocyte membrane. These compounds are returned to the intestinal lumen or released into portal circulation by intestinal efflux transporters expressed at apical or basolateral membranes, respectively. One important transporter superfamily, multiple members of which are intestinally expressed, are the solute carriers (SLCs). SLC expression levels may determine the pharmacokinetics of drugs that are substrates of these transporters. In this study we characterize the distribution of 15 human SLC transporter mRNAs in histologically normal biopsies from five regions of the intestine of 10 patients. The mRNA expression levels of CNT1, CNT2, apical sodium-dependent bile acid transporter (ABST), serotonin transporter (SERT), PEPT1, and OCTN2 exhibit marked differences between different regions of the intestine: the first five are predominantly expressed in the small intestine, whereas OCTN2 exhibits strongest expression in the colon. Two transporter mRNAs studied (OCTN1, OATP2B1) are expressed at similar levels in all gut sections. In addition, ENT2 mRNA is present at low levels across the colon, but not in the small intestine. The other six SLC mRNAs studied are not expressed in the intestine. Quantitative knowledge of transporter expression levels in different regions of the human gastrointestinal tract could be useful for designing intestinal delivery strategies for orally administered drugs. Furthermore, changes in transporter expression that occur in pathological states, such as inflammatory bowel disease, can now be defined more precisely by comparison with the expression levels measured in healthy individuals.

BACKGROUND & AIMS: Cholecystokinin (CCK) is a major gastrointestinal peptide hormone that is released postprandially from the small intestine and exerts marked effects on gallbladder and gastrointestinal motility. The smaller isoforms CCK-8 and CCK-4 are rapidly taken up into hepatocytes, metabolized, and excreted into bile. Our aim was to identify and characterize the hepatocellular CCK-8 uptake system. METHODS: CCK-8 uptake was measured in Xenopus laevis oocytes expressing the organic anion-transporting polypeptides of rat liver (Oatp1, Oatp2, Oatp3, or Oatp4) and of human liver (OATP-A, OATP-B, OATP-C, or OATP8) and in primary cultured rat hepatocytes. RESULTS: Rat Oatp4 and human OATP8 efficiently mediated CCK-8 uptake in oocytes, with Michaelis constant (Km) values of 14.9 +/- 2.9 micromol/L and 11.1 +/- 2.9 micromol/L, respectively. CCK-8 uptake by hepatocytes was also saturable, with a Km of 6.7 +/- 2.1 micromol/L. The Km value in rat hepatocytes is consistent with Oatp4-mediated transport. CONCLUSIONS: CCK-8 is selectively transported by rat Oatp4 and human OATP8, both of which are exclusively expressed at the basolateral membrane of hepatocytes. These 2 transporters are the first and probably the predominant hepatic uptake systems for CCK-8 and may be critical for the rapid clearance of this hormone from the circulation.

Background & Aims: Hepatic uptake of cholephilic organic compounds is mediated by members of the organic anion-transporting polypeptide (OATP) family. We aimed to characterize the novel OATP-B with respect to tissue distribution and hepatocellular localization and to compare its substrate specificity with those of OATP-A, OATP-C, and OATP8. Methods: Tissue distribution and hepatocellular localization of OATP-B were analyzed by Northern blotting and immunofluorescence, respectively. Transport of 16 substrates was measured for each individual human OATP in complementary RNA–injected Xenopus laevis oocytes. Results: Expression of OATP-B was most abundant in human liver, where it is localized at the basolateral membrane of hepatocytes. OATP-B, OATP-C, and OATP8 mediated high-affinity uptake of bromosulphophthalein (Km, ~0.7, 0.3, and 0.4 μmol/L, respectively). OATP-B also transported estrone-3-sulfate but not bile salts. Although OATP-A, OATP-C, and OATP8 exhibit broad overlapping substrate specificities, OATP8 was unique in transporting digoxin and exhibited especially high transport activities for the anionic cyclic peptides [d-penicillamine2,5]enkephalin (DPDPE; opioid-receptor agonist) and BQ-123 (endothelin-receptor antagonist). Conclusions: OATP-B is the third bromosulphophthalein uptake system localized at the basolateral membrane of human hepatocytes. OATP-B, OATP-C, and OATP8 account for the major part of sodium-independent bile salt, organic anion, and drug clearance of human liver. GASTROENTEROLOGY 2001;120:525-533

Sodium-dependent uptake of bile acids from blood is aliver-specific function which is mediated by theNa+-taurocholate cotransporting polypeptide(Ntcp). We report the stable expression of aNa+-taurocholate cotransporting green fluorescentfusion protein in the human hepatoblastoma cell lineHepG2, normally lacking Ntcp expression. Ntcp-EGFPassociated green fluorescence colocalized with Ntcpimmunofluorescence in the plasma membrane. Intransfected HepG2 cells, the fusion protein mediatedthe sodium-dependent uptake of the bile acidtaurocholate (Km: 24.6 μmol/l) and of the anionicsteroids estrone-3-sulfate and dehydroepiandrosteronesulfate. We conclude that the Ntcp-EGFP fusion proteinfollows the sorting route of Ntcp, is functionallyidentical to Ntcp and could be used to monitor proteintrafficking in living HepG2 cells.

Matrix metalloproteinases have been reported to be involved in tumor cell invasion and metastasis. Dissemination of malignant cells in acute myeloid leukemia (AML) may be mediated by similar mechanisms. Here, we report, that the t(15/17)+ acute promyelocytic leukemia (APL) cell line NB4 constitutively expresses and releases the proenzyme form of matrix metalloproteinase-9 (MMP-9, 92 kDa type IV collagenase/gelatinase, gelatinase B), as well as tissue inhibitor of metalloproteinases-1 (TIMP-1). Both proteins were identified by N-terminal amino acid sequence analysis after purification using gelatin Sepharose affinity chromatography. Whereas 12-O-tetradecanoylphorbol-13 acetate (TPA) increased both MMP-9 and TIMP-1 mRNA levels, tumor necrosis factor-alpha (TNF-alpha) stimulated only MMP-9 gene expression in a dose- and time-dependent manner. Neutralizing monoclonal antibodies (MoABs) to TNF-alpha (anti-TNF-alpha) decreased the constitutive and TPA-dependent expression of MMP-9 but did not influence TIMP-1 expression, either in unstimulated or in TPA-treated NB4 cells. FACS analyses showed that NB4 cells express both TNF receptor 1 (TNF-R1) and TNF-R2 to a similar extent. Blocking MoABs against TNF-R 1 (anti-TNF-R1) decreased the constitutive expression of MMP-9, whereas anti-TNF-R2 had almost no effect. Our results show, that in NB4 cells the expression of MMP-9 but not of TIMP-1 is maintained by autocrine stimulation with TNF-alpha. Thus, leukemic cells may be enabled to leave the bone marrow and infiltrate peripheral tissues by a dysfunction in the regulation of the MMP-9:TIMP-1 equilibrium, possibly triggered through autostimulation by TNF-alpha.

The pivotal role of organic anion-transporting polypeptide 1B1 (OATP1B1) in drug disposition has become clear over the last decade. Therefore, an OATP1B1 inhibition assay suitable for use within early drug discovery was developed and characterized. IC(50) estimates for 10 literature compounds using pitavastatin and estradiol-17β-glucuronide as substrates were within 2-fold of each other. In addition, the IC(50) estimates using pitavastatin uptake agreed well with literature values (r(2) = 0.92, average fold error = 1.3). However, when estrone-3-sulfate was used, OATP1B1 inhibition was underpredicted by as much as 10-fold. A comparison of uptake in human hepatocytes and OATP1B1 inhibition showed a significant correlation (r(2) = 0.53, P < 0.001) for more than 40 compounds. These data suggest that, for discrete chemical series, OATP1B1 inhibition data may be used as a surrogate for more costly and time-consuming uptake studies in hepatocytes. OATP1B1 inhibition data, determined for over 260 compounds representing both internal AstraZeneca and literature chemistry, were also used to generate a continuous in silico model. The robustness of the model was demonstrated by accurately predicting OATP1B1 inhibition for external test sets using 50 AstraZeneca compounds (root mean square error = 0.45) and 12 literature drugs (RMSE = 0.32). The most important molecular descriptors for the prediction of OATP1B1 inhibition were maximal hydrogen bonding strength followed by cLogP. This study has shown that a well validated OATP1B1 inhibition assay in conjunction with an in silico approaches has the potential to influence significantly the design-make-test cycle and subsequently reduce the propensity of OATP1B1 ligands.

The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. Conclusion: These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.

Muramyl peptides derived from bacterial peptidoglycan are detected intracellularly by Nod1 and Nod2, 2 members of the newly characterized nod-like receptor (NLR) family of pattern recognition molecules. In the absence of bacterial invasion into the host cytosolic compartment, it remains unclear whether muramyl peptides can cross the plasma membrane and localize into the cytosol. We have recently demonstrated that the plasma membrane transporter, hPepT1, was able to efficiently translocate muramyl dipeptide (MDP), a specific Nod2-activating molecule, into host cells. We aimed to characterize the transport properties of hPepT1 towards a spectrum of muramyl peptides, including Nod1-activating molecules. To do so, we designed an original procedure based on the ectopic expression of hPepT1 in oocytes from Xenopus laevis . Our results demonstrated that hPepT1 transports MDP but no other Nod2-activating molecule. Moreover, we observed that Nod1-stimulating muramyl peptides were not transported by hPepT1. Since hPepT1 expression is strongly associated with intestinal epithelial cells, where Nod1 and Nod2 have been shown to play a key role, these observations suggest a distinct contribution of Nod1 and Nod2 in mucosal homeostasis following the cellular uptake of muramyl peptides by hPepT1.

Background: Tegaserod is a novel drug for the treatment of constipation-predominant irritable bowel syndrome. Tegaserod is thought to exert its prokinetic effect as a selective partial agonist of serotonin receptor type 4 (5-HT4) receptors located in the enteric nervous system. It is unknown, however, whether tegaserod interacts with the human serotonin reuptake transporter (hSERT) and the uptake transporters for dopamine (hDAT) and norepinephrine (hNET). Therefore, the aim of the present study was to investigate whether tegaserod inhibits SERT-, DAT-, and NET-mediated transport. Methods: Tegaserod inhibition of SERT-mediated [3H]5-HT and NET- and DAT-mediated [3H]dopamine uptake was measured in human embryonic kidney (HEK) 293 cells stably expressing hSERT, hDAT, and hNET in comparison with untransfected control HEK293-FT cells. Results: Tegaserod inhibited SERT-, DAT-, and NET-mediated transport with IC50-values of 11.7, 20.7, and 3.2 µmol/l, respectively, while 100 µmol/l estrone-3-sulfate or taurocholic acid, used as negative controls, failed to inhibit hSERT-mediated transport. Using Dixon plot analysis, inhibition kinetics yielded a non-competitive type of inhibition with an apparent inhibition konstant (Ki) of 3.1 µmol/l for SERT-mediated 5-HT transport. Conclusion: In the present study we propose an additional mechanism of action for tegaserod as a serotonin uptake inhibitor. By inhibiting SERT and increasing local 5-HT concentrations in the gut wall, tegaserod might exert its prokinetic action via a synergism between 5-HT4 agonism and low-affinity SERT inhibition.

PURPOSE: To identify and localize the expression of multispecific organic anion transporting polypeptides (Oatps/OATPs) in the ciliary body epithelium and to investigate their possible involvement in the transport of the antiglaucoma agent unoprostone. METHODS: Oatps/OATPs were detected by immunoblot analysis and by immunofluorescence microscopy in homogenized and fixed rat and human ciliary body samples using specific polyclonal antibodies. Transport of 3H-labelled unoprostone was measured in Oatp/OATP expressing Xenopus laevis oocytes. RESULTS: Immunoblots of ciliary body extracts were positive for rat Oatp1a4, Oatp1a5 and Oatp1b2 and for human OATP1A2, OATP1C1, OATP2B1, OATP3A1 and OATP4A1. Confocal immunofluorescence microscopy localized Oatp1a4 and Oatp1b2 as well as all immunoblot positive human OATPs at the basolateral plasma membrane of the non-pigmented rat and human ciliary body epithelium, respectively. However, for human OATPs additional regional differences in expression were found with OATP1A2 and OATP1C1 being expressed only in the pars plana of human ciliary body epithelium. Furthermore, OATP1C1, OATP3A1 and OATP4A1 were also expressed at the basolateral plasma membrane of the pars plana pigmented epithelium. And finally, deesterified unoprostone carboxylate was found to be transported by OATP1A2, OATP2B1 and OATP4A1 with approximate K(m)-values of 93, 91 and 132 microm, respectively. CONCLUSIONS: Several multispecific organic anion transporting polypeptides are expressed at the basolateral plasma membrane of the non-pigmented, and to a lesser extent also of the pigmented, epithelium in rat and human ciliary body. These Oatps/OATPs can account for the previously suggested 'liver-like' transport functions of mammalian ciliary body epithelium.

Glycyrrhizin (GL) is used in Japan for the treatment of chronic hepatitis C. Following intravenous administration, GL is eliminated mainly by excretion into bile. Hepatocyte uptake of GL is a carrier-mediated process with characteristics resembling the organic anion transporting polypeptides (OATPs, solute carrier gene family SLC21A). We, therefore, studied whether GL is a potential transport substrate of the OATPs of rat and human liver. Because transport of GL could not be measured directly, GL-mediated cis-inhibition of [3H]estrone-3-sulfate or [35S]bromosulfophthalein uptake was analyzed kinetically in Xenopus laevis oocytes injected with cRNA coding for OATPs. GL inhibited [3H]estrone-3-sulfate uptake by 75–100% in oocytes expressing rat Oatp4, human OATP-C or human OATP8, members of the OATP1B subfamily that are expressed predominantly in hepatocytes. Dixon plots indicated a non-competitive type of inhibition, with Ki values of 6.1, 15.9 and 12.5 μmol/l, respectively. In contrast, GL inhibition of rat Oatp1, Oatp2 and Oatp3 and human OATP-A and OATP-B was only between 0 and 53%. In conclusion, GL is an inhibitor and, therefore, potentially a transport substrate of the liver-specific OATPs in rat and man. The rate at which GL is taken up into the liver may depend upon the function and expression levels of these hepatocellular OATPs.