The bile salt export pump

Canalicular secretion of bile salts mediated by the bile salt export pump Bsep constitutes the major driving force for the generation of bile flow. Bsep is a member of the B-family of the super family of ATP-binding cassette transporters and is classified as ABCB11. Bsep has a narrow substrate specificity, which is largely restricted to bile salts. Bsep is extensively regulated at the transcriptional and posttranscriptional level, which directly modulates canalicular bile formation. Pathophysiological alterations of Bsep by either inherited mutations or acquired processes such as inhibition by drugs or disease-related down regulation may lead to a wide spectrum of mild to severe forms of liver disease. Furthermore, many genetic variants of Bsep are known, some of which potentially render individuals susceptible to acquired forms of liver diseas

Transporters for Bile Formation in Physiology and Pathophysiology

The liver fulfills many vital functions for the body, among them bile formation and detoxification. Bile salts are organic anions, are the major constituents of bile and are at high concentrations cytotoxic. Detoxification exposes the liver to many harmful compounds. This function is therefore potentially damaging to the liver. Impaired bile formation may lead to hepatic accumulation of bile salts and subsequently to liver disease. Diagnosis of liver diseases involves the measurement of so-called liver function parameters. This overview aims to characterize and summarize the role of organic anion transporters in bile formation at the protein level under normal physiologic conditions and in liver function tests used for diagnosing liver diseases in pathophysiologic situations

Functional Identification of Arabidopsis ATSIP2 (At3g57520) as an Alkaline α-Galactosidase with a Substrate Specificity for Raffinose and an Apparent Sink-Specific Expression Pattern

Arabidopsis ATSIP2 has recently been suggested to be a raffinose synthase gene. However, it has high amino acid identity to functionally characterized alkaline α-galactosidases from Cucumis melo and Zea mays. Using the Sf9 insect cell expression system, we demonstrate that recombinant ATSIP2 is a genuine alkaline α-galactosidase with a distinct substrate specificity for raffinose, and not a raffinose synthase. A β-glucuronidase reporter construct using the ATSIP2 promoter shows that ATSIP2 is strongly expressed in sink tissues of Arabidopsis, i.e. sink leaves and non-xylem parts of the root stele, suggesting a physiological function in raffinose phloem unloadin

An Arabidopsis T-DNA Insertion Mutant for Galactokinase (AtGALK, At3g06580) Hyperaccumulates Free Galactose and is Insensitive to Exogenous Galactose

Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g−1 FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g−1 FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant backgroun

Effect of a Common Genetic Variant (p.V444A) in the Bile Salt Export Pump on the Inhibition of Bile Acid Transport by Cholestatic Medications

The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 μM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 μM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested

Structure of the human lipid exporter ABCB4 in a lipid environment

ABCB4 is an ATP-binding cassette transporter that extrudes phosphatidylcholine into the bile canaliculi of the liver. Its dysfunction or inhibition by drugs can cause severe, chronic liver disease or drug-induced liver injury. We determined the cryo-EM structure of nanodisc-reconstituted human ABCB4 trapped in an ATP-bound state at a resolution of 3.2 Å. The nucleotide binding domains form a closed conformation containing two bound ATP molecules, but only one of the ATPase sites contains bound Mg2+. The transmembrane domains adopt a collapsed conformation at the level of the lipid bilayer, but we observed a large, hydrophilic and fully occluded cavity at the level of the cytoplasmic membrane boundary, with no ligand bound. This indicates a state following substrate release but prior to ATP hydrolysis. Our results rationalize disease-causing mutations in human ABCB4 and suggest an 'alternating access' mechanism of lipid extrusion, distinct from the 'credit card swipe' model of other lipid transporters

Glucose Transporter 9 (GLUT9) Plays an Important Role in the Placental Uric Acid Transport System.

BACKGROUND Hyperuricemia is a common laboratory finding in pregnant women compromised by preeclampsia. A growing body of evidence suggests that uric acid is involved in the pathogenesis of preeclampsia. Glucose transporter 9 (GLUT9) is a high-capacity uric acid transporter. The aim of this study was to investigate the placental uric acid transport system, and to identify the (sub-) cellular localization of GLUT9. METHODS Specific antibodies against GLUT9a and GLUT9b isoforms were raised, and human villous (placental) tissue was immunohistochemically stained. A systemic GLUT9 knockout (G9KO) mouse model was used to assess the placental uric acid transport capacity by measurements of uric acid serum levels in the fetal and maternal circulation. RESULTS GLUT9a and GLUT9b co-localized with the villous (apical) membrane, but not with the basal membrane, of the syncytiotrophoblast. Fetal and maternal uric acid serum levels were closely correlated. G9KO fetuses showed substantially higher uric acid serum concentrations than their mothers. CONCLUSIONS These findings demonstrate that the placenta efficiently maintains uric acid homeostasis, and that GLUT9 plays a key role in the placental uric acid transport system, at least in this murine model. Further studies investigating the role of the placental uric acid transport system in preeclampsia are eagerly needed

Imaging techniques to study drug transporter function in vivo

Transporter systems involved in the permeation of drugs and solutes across biological membranes are recognized as key determinants of pharmacokinetics. Typically, the action of membrane transporters on drug exposure to tissues in living organisms is inferred from invasive procedures, which cannot be applied in humans. In recent years, imaging methods have greatly progressed in terms of instruments, synthesis of novel imaging probes as well as tools for data analysis. Imaging allows pharmacokinetic parameters in different tissues and organs to be obtained in a non-invasive or minimally invasive way. The aim of this overview is to summarize the current status in the field of molecular imaging of drug transporters. The overview is focused on human studies, both for the characterization of transport systems for imaging agents as well as for the determination of drug pharmacokinetics, and makes reference to animal studies where necessary. We conclude that despite certain methodological limitations, imaging has a great potential to study transporters at work in humans and that imaging will become an important tool, not only in drug development but also in medicine. Imaging allows the mechanistic aspects of transport proteins to be studied, as well as elucidating the influence of genetic background, pathophysiological states and drug-drug interactions on the function of transporters involved in the disposition of drugs

Effect of ritonavir on the pharmacokinetics of the benzimidazoles albendazole and mebendazole: an interaction study in healthy volunteers

Background: Benzimidazoles are often used concomitantly with protease inhibitors in patients with helminthic disease and HIV infection. Low bioavailability and extensive first-pass metabolism make benzimidazoles prone to pharmacokinetic drug interactions. The aim of the present study was to investigate potential drug interactions between the benzimidazoles albendazole and mebendazole and the potent CYP3A4 inhibitor ritonavir. Methods: Sixteen healthy volunteers were administered a single oral dose of 1,000mg mebendazole or 400mg albendazole (2 × n = 8). AUC, Cmax, and t1/2 of mebendazole, albendazole, and albendazole sulfoxide were studied in absence and after short-term (2 doses) and long-term (8days) treatment with ritonavir 200mg bid. Results: Pharmacokinetic parameters of albendazole and mebendazole were not changed by short-term administration of ritonavir. However, long-term administration of ritonavir resulted in significant changes in albendazole and mebendazole disposition, with a significant decrease in AUC0-24 (27 and 43% of baseline for albendazole and mebendazole, respectively) and Cmax (26 and 41% of baseline, respectively). Conclusion: The AUC0-24 of benzimidazoles decreased after long-term use of ritonavir, while no changes in pharmacokinetic profiles were observed under short-term administration. These findings might help to optimize benzimidazole efficacy when used in combination with protease inhibitor