Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human.

While P-glycoprotein (PGP, ABCB1) is known to play an important role in drug exclusion at the blood brain barrier (BBB), less is known about the contribution of other members in the ATP-binding cassette (ABC) transporter family to BBB drug efflux, or whether these transporters are expressed differently in humans and in mammalian species of pharmacological interest. We used quantitative real-time PCR to determine mRNA expression levels for the majority of ABC family members in brain and in isolated brain microvessel endothelial capillary cells (BMEC) from human, rat, mouse, pig and cow. We confirmed BBB expression of several well-characterized ABC family members that are implicated in xenobiotic exclusion from the brain, including ABCB1 (PGP), ABCG2 (BCRP), ABCC1 (MRP1), ABCC4 (MRP4), and ABCC5 (MRP5). In addition, we detected high expression and enrichment in BMEC of several less well-characterized ABC transporters in one or more species, including ABCA2-4, ABCB4, ABCB6-8, ABCB10, ABCC3, ABCC6, ABCC10, and ABCE1. We also uncovered species differences in the expression of a number of transporters, including ABCG2 and ABCC4. This study identifies several additional ABC family members that may contribute to xenobiotic efflux at the human BBB, and compares the expression of a broad array of efflux transporters between human and four other species relevant to pharmacological research.

Expression of the thyroid hormone transporters monocarboxylate transporter-8 (SLC16A2) and organic ion transporter-14 (SLCO1C1) at the blood-brain barrier.

Thyroid hormones require transport across cell membranes to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human monocarboxylate transporter-8 (MCT8) (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that organic ion transporter polypeptide-14 (OATP14, also known as OATP1C1/SLCO1C1) is the primary thyroid hormone transporter at the blood-brain barrier, whereas MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein are strongly enriched in mouse and rat cerebral microvessels but not in human microvessels. In rat, Mct8 and Oatp14 proteins localize to both the luminal and abluminal microvessel membranes. In human and rodent choroid plexus epithelial cells, MCT8 is concentrated on the epithelial cell apical surface and OATP14 localizes primarily to the basal-lateral surface. Mct8 and Oatp14 expression was also observed in mouse and rat tanycytes, which are thought to form a barrier between hypothalamic blood vessels and brain. These results raise the possibility that reduced thyroid hormone transport across the blood-brain barrier contributes to the neurological deficits observed in affected patients with MCT8 mutations. The high microvessel expression of OATP14 in rodent compared with human brain may contribute to the relatively mild phenotype observed in Mct8-null mice, in contrast to humans lacking functional MCT8.

Regulation of the sodium/sulfate co-transporter by farnesoid X receptor alpha.

Fxralpha is known to regulate a variety of metabolic processes, including bile acid, cholesterol, and carbohydrate metabolism. In this study, we show direct evidence that Fxralpha is a key player in maintaining sulfate homeostasis. We identified and characterized the sodium/sulfate co-transporter (NaS-1; Slc13a1) as an Fxralpha target gene expressed in the kidney and intestine. Electromobility shift assays, chromatin immunoprecipitation, and promoter reporter studies identified a single functional Fxralpha response element in the second intron of the mouse Slc13a1 gene. Treatment of wild-type mice with GW4064, a synthetic Fxralpha agonist, induced Slc13a1 mRNA in the intestine and kidney. Slc13a1 mRNA was also induced in the kidney and intestine of wild-type, but not Fxralpha-/- mice, after treatment with the hepatotoxin alpha-naphthylisothiocyanate, which is known to result in elevated blood bile acid levels. Finally, we observed a decrease in Slc13a1 mRNA in the kidney and intestine of Fxralpha-/- mice and a corresponding increase in urinary excretion of free sulfates as compared with wild-type mice. These results demonstrate that mouse Slc13a1 is a novel Fxralpha target gene expressed in the kidney and intestine and that in the absence of Fxralpha, mice waste sulfate into the urine. Thus, Fxralpha is necessary for normal sulfate homeostasis in vivo.

The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter.

Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)alpha-Ostbeta. By Northern blot analysis, Ostalpha and Ostbeta mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostalpha and Ostbeta protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostalpha-Ostbeta were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostalpha-Ostbeta, but not the individual subunits, stimulated Na(+)-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostalpha-Ostbeta expression. Co-expression of Ostalpha and Ostbeta was required to convert the Ostalpha subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostalpha through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostalpha and Ostbeta. These results demonstrate that the mouse Ostalpha-Ostbeta heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.

XP13512 [(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane acetic acid], a novel gabapentin prodrug: I. Design, synthesis, enzymatic conversion to gabapentin, and transport by intestinal solute transporters.

Gabapentin is thought to be absorbed from the intestine of humans and animals by a low-capacity solute transporter localized in the upper small intestine. Saturation of this transporter at doses used clinically leads to dose-dependent pharmacokinetics and high interpatient variability, potentially resulting in suboptimal drug exposure in some patients. XP13512 [(+/-)-1-([(alpha-isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane acetic acid] is a novel prodrug of gabapentin designed to be absorbed throughout the intestine by high-capacity nutrient transporters. XP13512 was stable at physiological pH but rapidly converted to gabapentin in intestinal and liver tissue from rats, dogs, monkeys, and humans. XP13512 was not a substrate or inhibitor of major cytochrome P450 isoforms in transfected baculosomes or liver homogenates. The separated isomers of XP13512 showed similar cleavage in human tissues. The prodrug demonstrated active apical to basolateral transport across Caco-2 cell monolayers and pH-dependent passive permeability across artificial membranes. XP13512 inhibited uptake of (14)C-lactate by human embryonic kidney cells expressing monocarboxylate transporter type-1, and direct uptake of prodrug by these cells was confirmed using liquid chromatography-tandem mass spectrometry. XP13512 inhibited uptake of (3)H-biotin into Chinese hamster ovary cells overexpressing human sodium-dependent multivitamin transporter (SMVT). Specific transport by SMVT was confirmed by oocyte electrophysiology studies and direct uptake studies in human embryonic kidney cells after tetracycline-induced expression of SMVT. XP13512 is therefore a substrate for several high-capacity absorption pathways present throughout the intestine. Therefore, administration of the prodrug should result in improved gabapentin bioavailability, dose proportionality, and colonic absorption compared with administration of gabapentin.

Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes.

To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel. Rescuing the folding defect or down-regulating the E1 ubiquitin (Ub)-activating enzyme stabilized the mutant CFTR without interfering with its internalization. These observations with the preferential association of mutant CFTRs with Hrs, STAM-2, TSG101, hVps25, and hVps32, components of the Ub-dependent endosomal sorting machinery, establish a functional link between Ub modification and lysosomal degradation of misfolded CFTR from the cell surface. Our data provide evidence for a novel cellular mechanism of CF pathogenesis and suggest a paradigm for the quality control of plasma membrane proteins involving the coordinated function of ubiquitination and the Ub-dependent endosomal sorting machinery.

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.

Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart.

G protein-activated inwardly rectifying potassium channels (Kir3, GIRK) provide an important mechanism for neurotransmitter regulation of membrane excitability. GIRK channels are tetramers containing various combinations of Kir3 subunits (Kir3.1--Kir3.4). We find that different combinations of Kir3 subunits exhibit a surprisingly complex spectrum of trafficking phenotypes. Kir3.2 and Kir3.4, but not Kir3.1, contain ER export signals that are important for plasma membrane expression of Kir3.1/Kir3.2 and Kir3.1/Kir3.4 heterotetramers, the GIRK channels found in the brain and the heart, respectively. Additional motifs in Kir3.2 and Kir3.4 control the trafficking between endosome and plasma membrane. In contrast, the Kir3.3 subunit potently inhibits plasma membrane expression by diverting the heterotetrameric channels to lysosomes. Such rich trafficking behaviors provide a mechanism for dynamic regulation of GIRK channel density in the plasma membrane.