Organic Anionic Compounds Research Articles

Role of Organic Anion Transporter NPT4 in Renal Handling of Uremic Toxin 3-indoxyl Sulfate

Sodium-phosphate transporter NPT4 (SLC17A3) is a membrane transporter for organic anionic compounds localized on the apical membranes of kidney proximal tubular epithelial cells and plays a role in the urinary excretion of organic anionic compounds. However, its physiological role has not been sufficiently elucidated because its substrate specificity is yet to be determined. The present study aimed to comprehensively explore the physiological substrates of NPT4 in newly developed Slc17a3−/− mice using a metabolomic approach. Metabolomic analysis showed that the plasma concentrations of 11 biological substances, including 3-indoxyl sulfate, were more than two-fold higher in Slc17a3−/− mice than in wild-type mice. Moreover, urinary excretion of 3-indoxyl sulfate was reduced in Slc17a3−/− mice compared to that in wild-type mice. The uptake of 3-indoxyl sulfate by NPT4-expressing Xenopus oocytes was significantly higher than that by water-injected oocytes. The calculated Km and Vmax values for NPT4-mediated 3-indoxyl sulfate uptake were 4.52 ± 1.18 mM and 1.45 ± 0.14 nmol/oocyte/90 min, respectively. In conclusion, the present study revealed that 3-indoxyl sulfate is a novel substrate of NPT4 based on the metabolomic analysis of Slc17a3−/− mice, suggesting that NPT4 regulates systemic exposure to 3-indoxyl sulfate by regulating its urinary excretion.

Evaluation of Large Organic Anion Transport at the Blood‐CSF Barrier by Quantitative Fluorescence Microscopy

The blood-cerebrospinal fluid barrier (BCSFB), formed by the epithelial cells of the choroid plexus, expresses several multispecific membrane transporters that actively remove drugs, toxins, and metabolic wastes from the brain. Transporter-mediated clearance by the BCSFB typically occurs as a two-step process, where uptake transporters on the apical membrane extracts drug from the CSF into the choroid plexus cells, and efflux transporters on the basolateral membrane extrude intracellular drug into the blood. These transporters can be determinants of local cerebrospinal fluid (CSF) drug concentrations, impacting drug efficacy, local toxicity, or drug interaction predictions within the CNS. However, transporter activity at the BCSFB is understudied and remains poorly characterized. The objective of this study is to develop and utilize a quantitative fluorescent microscopy approach to evaluate organic anion transport mediated by organic anion transporting peptide (Oatp) at the murine BCSFB. Confocal imaging was used to observe time-dependent uptake of several large organic anion fluorescent probes in live choroid plexus tissue isolated from adult C57BL/6 mice. Image segmentation was performed using ImageJ to individually quantify intracellular choroid plexus cell and blood capillary accumulation. The fluorescent probes fluorescein-methotrexate (FL-MTX), fluorescein-cyclicAMP (Fluo-cAMP), and sulforhodamine101 (SR101) all demonstrated rapid transepithelial transport across the BCSFB from the CSF into the blood, reaching a peak accumulation within 15 minutes. Choroid plexus cell accumulation remains minimal, suggesting apical uptake as the rate limiting step for transepithelial BCSFB transport of large organic anions. FL-MTX, Fluo-cAMP, and SR101 accumulation were abolished by rifampin or bromosulfophthalein inhibition. Both rifampin and bromosulfophthalein are classical OATP/Oatp inhibitors, indicating functional contribution of OATP/Oatps for apical uptake at the BCSFB. MK571, a potent MRP inhibitor, increased choroid plexus cell accumulation and reduced blood capillary accumulation, consistent with MRP function at the basolateral membrane. This work suggests that Oatps at the murine BCSFB are capable of actively and rapidly removing a range of large organic anion compounds from the CSF, and that apical uptake is the rate limiting step for transport of large organic anions across the BCSFB.

Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4

To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

Evaluation of [18F]pitavastatin as a positron emission tomography tracer for in vivo organic transporter polypeptide function

IntroductionTo understand the pathways involved in drug clearance from the body, quantitative evaluations of the hepatobiliary transport of drugs are important. The organic anion transporting polypeptide (OATP) family transporter, particularly OATP1B1 and 1B3, are considered to play an important role in hepatic uptake of organic anion compounds. Pitavastatin is a substrate of OATP, and it includes a fluorine group. Therefore, it represents an acceptable positron-emission tomography (PET) tracer using fluorine-18 to image in vivo hepatic transporter functions. Method[18F]Pitavastatin was synthesized using the method we previously reported. To evaluate the potential of [18F]pitavastatin in PET imaging of in vivo OATP functions, we investigated the hepatic uptake with/without rifampicin as an OATP inhibitor after administration in normal SD rats. [18F]Pitavastatin metabolite was evaluated using reverse-phase thin-layer chromatography (TLC) autoradiography. We subsequently analyzed the PET image results and demonstrated that [18F]pitavastatin selectively accumulated in the liver post-administration.Result and discussionIn metabolite analysis using reverse-phase TLC, we found that the radioactivity detected in the plasma, liver (>90% intact), and bile mostly originated from the parent pitavastatin of the PET study (~40 min). [18F]pitavastatin's hepatic uptake decreased (approximately 76%) with rifampicin co-administration in PET analysis. Because [18F]pitavastatin has lower clearance in rats than other previously reported OATP1B PET s, it holds the potential of an imaging tracer that has a higher sensitivity in monitoring hepatic OATP1B function's changes. ConclusionCompared with the previously reported OATP imaging tracers, [18F]pitavastatin is more suitable for the sensitive detection of functional changes in OATP transporters. We believe that [18F]pitavastatin enables quantitative analysis of the hepatobiliary transport system for organic anion compounds.

Organic anion-transporting polypeptide 1a4–mediated heterogeneous distribution of sulforhodamine-101 in rat hepatic lobules

It has been known that organic anion-transporting polypeptides (Oatps) involve hepatic transports several organic anionic compounds and drugs. This study aimed to investigate sulforhodamine-101 (SR-101) distribution in the rat liver, determine the molecules responsible for the distribution, and delineate the manner of distribution. After intravenous SR-101 administration, its distribution in frozen rat hepatic sections was examined. SR-101-derived signals were detected in regions around the hepatic central vein (CV), where immunohistochemistry (IHC) indicated high Oatp1a4 expression. The signals decreased with treatment by digoxin, a specific substrate for Oatp1a4. In vitro studies using isolated rat hepatocytes and rat Oatp1a4-expressing Xenopus laevis oocytes have suggested that SR-101 is an Oatp1a4 substrate and is taken up into rat hepatocytes mainly via Oatp1a4. Therefore, results suggested SR-101 zonation because of Oatp1a4 involvement and that Oatp1a4 function is dominant in the region around the hepatic CV in rat hepatic lobules.

A Putative Role of Organic Anion Transporting Polypeptides (Oatps) In Cell Survival of Hormone-Dependent Breast and Prostate Cancers

Transporter proteins classified into the solute carrier (SLC) transporter superfamily are essential for import of nutrients for cell survival in organisms. In the last two decades, compelling evidence has accumulated that SLC transporters interact with clinically important anticancer agents and contribute to their pharmacokinetics, particularly the biopharmaceutical processes of absorption, elimination and distribution. Furthermore, many SLC transporters have been shown to be differentially upregulated in cancer cells, and this may represent an adaptive response to altered nutritional requirements. Thus, it is likely to utilize them as carrier for efficient drug delivery as well as pharmacological target to shut off the essential nutrients for cell growth of malignant tumors. This short review will introduce organic anion transporting polypeptides which recognize endo- and exogenous organic anionic compounds and recent findings about their upregulation in cancer cells. Besides, OATP-mediated sulfate conjugates of steroid hormone may contribute to cell survival and adapted growth under hormonedepleted conditions. Better understandings of pathophysiological role of OATPs likely provide key information to overcome hormone-refractory breast and prostate tumors.

Modifying the surface of TEOS xerogel by metal ion Zn(II)

The removal of 2,4-dichlorophenol from aqueous solution by modified TEOS xerogel has been studied in batch experiments at varying pH (2.0–8.0). Tetraethyl orthosilicate (TEOS) xerogel recently has been considered as an adsorbent which is more active. Surface of the adsorbent due to the outward oxygen atoms in the molecular structure, is negatively charged, so this solid, shows good ability to adsorb metal ions. But because of the negative charge on the surface of the adsorbent, the anions, particularly organic anion compounds are not absorbed. To fix this problem, it can set the metal ions on the surface of the solid. By placing Zn ions on the xerogel, a positive charge on the surface of the adsorbent is made, which increases the ability of the adsorbent to attract 2, 4-dichlorophenol from aqueous solutions. Results show an increase in the adsorption by about 17% on empty xerogel, by about 60% in xerogel modified with metal Zn (II) ion.

Insulin stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1 in the human intestinal cell line Caco-2

Organic anion transporting polypeptide 2B1 (OATP2B1) is the major uptake transporter in the intestine, and transports various clinically used therapeutic agents. Insulin acts through the insulin receptor in targeted cells, and Rab8A is one of the insulin signaling pathways. The small intestine in humans also expresses insulin receptor and Rab8A. It has been reported that insulin stimulates peptide transporter 1 (PEPT1) expression at the apical membrane and increases uptake of PEPT1 substrates in small intestine epithelial model cells (Caco-2 cells). However, the effect of insulin on OATP2B1 in the small intestine has not been fully investigated. We found that Rab8A was associated with OATP2B1-mediated estrone-3-sulfate (E3S) uptake. Insulin stimulated the uptake of E3S by Caco-2 cells and the enhancement was sustained for 120 min. The Vmax value of E3S uptake significantly increased upon insulin exposure. Caco-2 cells treated with insulin showed increased OATP2B1 expression at the cell surface. The apical-to-basal transport of E3S was also increased by insulin. The increase of E3S transport was inhibited by the cold condition (4 °C) or the OATP2B1 inhibitor, taurocholate. These results indicate that insulin acts on the small intestine to increase OATP2B1-mediated absorption.

Quercetin‐3‐rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1

Quercetin-3-rhamnoglucoside (rutin) has a wide spectrum of biochemical and pharmacological activities. Rutin is absorbed mainly in its unmetabolized form. Organic anion transporting polypeptide (OATP) 2B1 is a major uptake transporter in the intestine. Thus, it is important for the prevention of adverse events to understand drug interactions mediated by OATP2B1 in the absorption process. This study assessed the effect of rutin on transport by OATP2B1. Rutin stimulated the uptake of estrone-3-sulfate (E-3-S), taurocholic acid (TCA), cholic acid (CA) and rosuvastatin by OATP2B1, but not p-coumaric acid or ferulic acid. The EC50 of rutin for transport by OATP2B1 was 2.32 μm. The Km value of E-3-S for OATP2B1 in the presence of rutin (9.21 μm) was almost the same as that in the absence of rutin (8.53 μm). On the other hand, the Vmax of E-3-S transport by OATP2B1 in the presence of rutin (270 pmol/mg protein/min) was 1.2-fold higher than that in the absence of rutin (218 pmol/mg protein/min). Moreover, the expression level of OATP2B1 on the cell membrane was increased by treatment with rutin for 5 min without alteration of the total OATP2B1 expression level. Moreover, the increase in the localization of OATP2B1 at the cell surface was detected by the immunocytochemistry. The stimulatory effect of rutin is a little weak but may affect the absorption of OATP2B1 substrates, because rutin is taken daily in foods and its intestinal concentration would reach the stimulatory range of OATP2B1.

Rapid Stimulating Effect of the Antiarrhythmic Agent Amiodarone on Absorption of Organic Anion Compounds

In a clinical setting, changes in pharmacokinetics due to drug-drug interactions can often directly affect the therapeutic safety and efficacy of drugs. Recently, interest has been shown in drug-drug interactions in the intestine. It is now recognized that changes in the functions of drug transporters substantially influence the absorption of administered drugs from the intestine. Amiodarone (AMD) is a potent drug used in the treatment of serious supraventricular and ventricular tachyarrhythmias. Despite its potent pharmacological effects, its wide clinical use is precluded by drug-drug interactions. In this study, we characterized the transporter function between AMD and various compounds in human intestinal model Caco-2 cells. AMD significantly and rapidly increased the uptake of [(3)H]estrone-3-sulfate (E-3-S) for 5 min. The apical-to-basal transport of [(3)H]E-3-S was significantly increased by AMD. The AMD-stimulated [(3)H]E-3-S uptake was inhibited by organic anion transporting polypeptide (OATP) substrates. Caco-2 cells treated with AMD showed increased OATP2B1 expression on the cell surface. AMD also increased the absorption of sulfobromophthalein (BSP), which is a typical organic anion compound, and the expression level of Oatp2b1 at the membrane in in vivo experiments. The results indicate that AMD induces OATP2B1/Oatp2b1 expression at the membrane in the intestine and enhances absorption of organic anion compounds.

Interactions Between Organic Anions on Multiple Transporters in Caco-2 Cells

In drug development, Caco-2 cells are often employed to study the influence of membrane transporters on drug permeability. The aim of the current study was to characterize permeability and kinetic parameters of selected organic anionic compounds in Caco-2 cells, and to investigate whether the Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide. The effects of these compounds on initial uptake, apparent permeability, and intracellular end-point accumulations of the probe substrates were studied. Both interactions on apical and basolateral influx transporters were observed. These interactions were proposed to be mediated mainly by the apical sodium-dependent bile acid transporter and the organic solute transporter α/β, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may be the key to further distinguish between interactions on multiple transporters, including the involvement of efflux transporters, by taking into account the changes in dynamics that drug interactions may cause. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3817–3830, 2011

The Impact of Hepatic Uptake on the Pharmacokinetics of Organic Anions

The disposition of seven marketed and two AstraZeneca acid (organic anion) compounds with a range of volume of distribution at steady state (V(ss)) and clearance have been profiled in rat and dog. Pharmacokinetic (PK) parameters along with liver and muscle tissue levels were collected, and their contributions to total V(ss) were calculated. The physiologically based prediction of V(ss) correlated (all predictions within 2-fold) with the V(ss) obtained from plasma PK analysis. The V(ss) of the acid drugs with atypically high values could be explained by significant sequestering of compound to the liver. A "media loss" in the in vitro hepatocyte assay that monitors loss of compound from the incubation media along with physiologically based PK (PBPK) modeling was assessed for its ability to accurately predict the impact of hepatic uptake on both clearance and V(ss). This methodology significantly improved the prediction of metabolic in vivo clearance compared with standard hepatocyte scaling approaches that do not take into account hepatic uptake. Predictions of V(ss) from the media loss assay also correlate with the measured values from plasma PK analysis. However, hepatic uptake will have little overall impact on half-life, because of the concomitant impact on both Cl and V(ss), as long as hepatic extraction is not high. The methodology described here is particularly useful when there is no allometric relationship between species as a result of interspecies differences in liver uptake. In this situation, the potential use of human hepatocytes combined with PBPK modeling avoids the question of which species pharmacokinetics is most predictive to humans.

Selection of Separation Conditions for HPLC and HPLC/MS of Aromatic Sulphonic Acids and Acid Azo Dyes

Liquid chromatography of organic anionic compounds usually cannot be performed without ionic additives to the mobile phase. Buffered mobile phases usually do not provide sufficient suppression of the ionization of strong acids, such as anionic dyes and aromatic sulfonic acids used in the dye production, or as whitening agents or detergents in various industrial and household applications. Hence, other modes are usually necessary for successful separation of these compounds, including ion‐exchange chromatography, ion‐interaction reversed‐phase chromatography, or ion‐pair chromatography. The retention mechanisms in these modes are still a controversial issue. In the present work, various theoretical models describing the retention of strong organic acids with bulky hydrophobic structural elements are reviewed. Conventional ionic additives employed in these separation modes usually are not volatile enough for HPLC/MS applications, such as quaternary ammonium salts commonly used as ion‐pairing reagents in ion‐pair chromatography. This inconvenience can be overcome either by removing the nonvolatile additives from the mobile phase before introducing the column effluent to the ion source of the mass spectrometer, or by using more volatile compounds as their substitution. Suitable volatile reagents should be selected taking into account their effects on the retention, separation selectivity, efficiency, and band symmetry of various compounds differing in structures. Another important point that should be considered in method development is possible suppression of ionization by the additives, which should be avoided.

Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.

Apical/Basolateral Surface Expression of Drug Transporters and its Role in Vectorial Drug Transport

It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug-drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood-tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin-Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.

Eisai Hyperbilirubinemic Rat (EHBR) as an Animal Model Affording High Drug-Exposure in Toxicity Studies on Organic Anions

The Eisai hyperbilirubinemic rat (EHBR) should be a useful animal model for studies on the toxicity of organic anions which are substrates of multidrug resistance-associated protein 2 (Mrp2), since the systemic exposure to these compounds is expected to be increased in EHBR. In this study, we tested the value of EHBR for this purpose, using pravastatin (PV) and methotrexate (MTX) as model compounds. In the case of a single oral dose of PV (200 mg/kg), C(max) in plasma was 4.0-fold higher and AUC(0-infinity) was 3.6-fold larger than those of normal Sprague-Dawley rats (SDR), respectively. When multiple doses of PV were given to EHBR without co-administration of any other compound, drug-induced skeletal muscle toxicity (myopathy/rhabdomyolysis) and increased creatine phosphokinase (CPK) level were observed, whereas a control experiment using SDR did not show any toxic change. When a single dose of MTX (0.6 mg/kg) was given to EHBR orally, C(max) was 1.7-fold higher and AUC(0-infinity) was 1.6-fold larger than those of SDR, respectively. When multiple doses of MTX were given to EHBR, the changes in bone marrow, spleen and intestines were more severe than those in SDR. These findings support the view that EHBR would be a valuable animal model for toxicity studies on organic anion compounds which are substrates of Mrp2.

Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/ SLCO superfamily, new nomenclature and molecular/functional properties.

The organic anion transporting polypeptides (rodents: Oatps, human: OATPs) form a superfamily of sodium-independent transport systems that mediate the transmembrane transport of a wide range of amphipathic endogenous and exogenous organic compounds. Since the traditional SLC21 gene classification does not permit an unequivocal and species-independent identification of genes and gene products, all Oatps/OATPs are newly classified within the OATP/ SLCO superfamily and subdivided into families (>/=40% amino acid sequence identity), subfamilies (>/=60% amino acid sequence identity) and individual genes and gene products according to their phylogenetic relationships and chronology of identification. Implementation of this new classification and nomenclature system occurs in agreement with the HUGO Gene Nomenclature Committee (HGNC). Among 52 members of the OATP/ SLCO superfamily, 36 members have been identified so far in humans, rat and mouse. The latter are clustered within 6 (out of 12) families (OATP1-OATP6) and 13 subfamilies. Oatps/OATPs represent 12 transmembrane domain proteins and contain the superfamily signature D-X-RW-(I,V)-GAWW-X-G-(F,L)-L. Although species divergence, multispecificity and wide tissue distribution are common characteristics of many Oatps/OATPs, some members of the OATP/ SLCO superfamily are highly conserved during evolution, have a high substrate specificity and exhibit unique cellular expression in distinct organs. Hence, while Oatps/OATPs with broad substrate specificity appear to play an important role in the bioavailability, distribution and excretion of numerous exogenous amphipathic organic anionic compounds, Oatps/OATPs with a narrow spectrum of transport substrates may exhibit more specific physiological functions in distinct organs.

Mechanism of lonidamine inhibition of the CFTR chloride channel.

1. The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is blocked by a broad range of organic anionic compounds. Here we investigate the effects of the indazole compound lonidamine on CFTR channels expressed in mammalian cell lines using patch clamp recording. 2. Application of lonidamine to the intracellular face of excised membrane patches caused a voltage-dependent block of CFTR currents, with an apparent K(d) of 58 micro M at -100 mV. 3. Block by lonidamine was apparently independent of channel gating but weakly sensitive to the extracellular Cl(-) concentration. 4. Intracellular lonidamine led to the introduction of brief interruptions in the single channel current at hyperpolarized voltages, leading to a reduction in channel mean open time. Lonidamine also introduced a new component of macroscopic current variance. Spectral analysis of this variance suggested a blocker on rate of 1.79 micro M(-1) s(-1) and an off-rate of 143 s(-1). 5. Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) significantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. 6. These results identify and characterize lonidamine as a novel CFTR open channel blocker and provide important information concerning its molecular mechanism of action.

Role of MRP family proteins in the export of organic anion compounds from liver and intestine

MRP and cMOAT are the organic anion transporters involved in the efflux of organic anions from cells. We have already found two novel cDNA sequences (MLP-1 and -2: MRP like protein) which show high homology with MRP and cMOAT. Liver expression of MLP-2 was observed only in mutant rats (EHBR) whose cMOAT is hereditarily defective, suggesting that MLP-2 is an inducible transporter. MLP-2 is induced in normal rat liver by injection of phenobarbital, ANIT and bile duct ligation. In normal rats, MLP-2 is expressed mainly in the duodenum and jejunum. To clarify the nature of the organic anion transport mechanism in the intestine, we used Caco-2 cells as an intestinal model. DNP-SG and estradiol 17 β-D-glucuronide were taken up by BBMV prepared from Caco-2 in an ATP-dependent manner. Northern blot analysis suggested high expression of human cMOAT and MLP-2 and low expression of MRP in cultured Caco-2 cells.

Interfacing ion chromatography with particle beam mass spectrometry for the determination of organic anionic compounds

In this paper we demonstrate the successful interfacing of ion chromatography with particle beam mass spectrometry through the use of a membrane suppressor for continuous desalting. The microbore version of the membrane suppressor does not introduce significant peak broadening. The detection limit for six aromatic sulfonic acids in the full scan mode is better than 0.4 μg injected on-column