Mediated Transport Research Articles

Monocarboxylate 4 Mediated Butyrate Transport in a Rat Intestinal Epithelial Cell Line

Short chain fatty acids (SCFA) are absorbed by carrier mediated uptake in the small intestine by pH-dependent SCFA/HCO3 (-) exchangers on the apical membrane of epithelial cells. Conventional assumption is that MCT1 mediates SCFA/HCO3 (-) exchange in the intestine. Further, due to the presence of multiple such anion exchangers, the identity of the intestinal SCFA/HCO3 (-) has been controversial. The aim of this study was to determine the identities of the butyrate transporter in the intestinal epithelial cells (IEC-18). IEC-18 cells were treated with specific siRNAs for MCT1 and MCT4, and butyrate and lactate uptake studies were performed. Alpha-cyano-4-hydroxycinnamic acid inhibited lactate uptake but not butyrate uptake in IEC-18 cells, indicating that these two substrates are transported via two different transporter systems. MCT1 siRNA treatment abolished both MCT1 mRNA by more than 95% and protein expression by 83% as evidenced by RTQ-PCR and western blotting experiments. However, MCT1 siRNA treatment inhibited butyrate uptake upto 24%, whereas it inhibited lactate uptake significantly by 70%. Treatment with MCT4 siRNA inhibited MCT4 mRNA expression by 75% and protein expression by 85% in these cells. MCT4 siRNA inhibited butyrate uptake by 40%. Further, several non-steroidal anti-inflammatory drugs (NSAIDs) are transported by the butyrate transporter. Finally, MCT4 siRNA inhibited salicylate uptake by 27% indicating direct evidence for the transport of salicylate by MCT4. These data indicate that MCT1 is the high affinity lactate transporter and MCT4 is the high affinity butyrate transporter in the intestinal epithelial cell line IEC-18.

P-glycoprotein Inhibition for Optimal Drug Delivery

P-glycoprotein (P-gp), an efflux membrane transporter, is widely distributed throughout the body and is responsible for limiting cellular uptake and the distribution of xenobiotics and toxic substances. Hundreds of structurally diverse therapeutic agents are substrates to it and it impedes the absorption, permeability, and retention of the drugs, extruding them out of the cells. It is overexpressed in cancer cells and accountable for obstructing cell internalization of chemotherapeutic agents and for developing transporter mediated resistance by cancer cells during anti-tumor treatments. As it jeopardizes the success of drug delivery and cancer targeting, strategies are being developed to overcome P-gp mediated drug transport. This concise review represents a brief discussion on P-gp mediated drug transport and how it hinders the success of various therapies. Its main focus is on various strategies used to tackle this curb in the field of drug delivery and targeting.

Water Mediated Membrane Transport: From Channels to Proton Pumps

Molecular simulations provide a powerful tool to study the energetics, dynamics, and mechanisms of biological membrane transport. We have used molecular dynamics simulations and modeling to study the gated transport of ions through transmembrane channels in signal transduction and the uphill pumping of protons in biological energy transduction. Remarkably, water consistently emerges as a critical player in the function of these molecular machines. In my talk I will examine how proteins exploit the unique properties of water at the nanoscale to achieve both high efficiency and high fidelity in a range of membrane transport phenomena.

Carrier Mediated Transport of Thorium from Nitric Acid Medium using 2-Ethyl Hexyl Hydrogen 2-Ethyl Hexyl Phosphonate (PC88A)/N-Dodecane as Carrier

The present study deals with the application of supported liquid membrane (SLM) technique for the separation of thorium from nitric acid medium using 2-ethyl hexyl hydrogen 2-ethyl hexyl phosphonate (PC88A) as a carrier and aqueous ammonium carbonate as a receiving phase. The effects of feed acidity, nature of strippant, and membrane pore size and membrane thickness on the transport of thorium have been studied in detail. Transport behavior of uranium (233U) and fission products from a radioanalytical laboratory waste is also studied. Stability of the membrane against the leaching of the extractant and stability of the membrane support have been investigated. An attempt has been made to model the physicochemical transport of thorium in SLM and establish the mechanism of thorium transport. Transport of thorium increased from 25% to about 96% using 0.75 M PC88A in n-dodecane as carrier and 2 M ammonium carbonate as stripping phase as the feed acidity decreased from 4 M HNO3 to 0.5 M HNO3. Optimum conditions obtained from this study were applied to recover thorium and 233U from analytical waste generated in the laboratory.

Potential Role of Sodium-Proton Exchangers in the Low Concentration Arsenic Trioxide-Increased Intracellular pH and Cell Proliferation

Arsenic main inorganic compound is arsenic trioxide (ATO) presented in solution mainly as arsenite. ATO increases intracellular pH (pHi), cell proliferation and tumor growth. Sodium-proton exchangers (NHEs) modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK) cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0–48 hours) in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5–100 µmol/L, NHEs inhibitor), PD-98059 (30 µmol/L, MAPK1/2 inhibitor), Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor), or Schering 28080 (10 µmol/L, H+/K+ATPase inhibitor) plus concanamycin (0.1 µmol/L, V type ATPases inhibitor). Incorporation of [3H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na+-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44mapk) were also determined. Lowest ATO (0.05 µmol/L, ∼0.01 ppm) used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1)-like transport dependent-increased pHi requiring p42/44mapk and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and in circumstances where ATO, likely arsenite, is available at the drinking-water at these levels.

Intestinal perfusion indicates high reliance on paracellular nutrient absorption in an insectivorous bat Tadarida brasiliensis

Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75–180Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (l-proline, MW 115Da, and d-glucose, MW 180Da) and two carbohydrates that have no mediated transport (l-arabinose, MW 150Da, and lactulose, MW 342Da). Absorption of lactulose (0.61±0.06nmolmin−1cm−1) was significantly lower than that of the smaller arabinose (1.09±0.04nmolmin−1cm−1). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10mM and 75mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1±3.0% to 66.2±7.8% of proline absorption is not transporter-mediated (varying proline from 1mM to 75mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.

Effect of salt concentration and mediators in salt bridge microbial fuel cell for electricity generation from synthetic wastewater

The aim of this study was to investigate the feasibility of using agar salt bridges for proton transport in Microbial Fuel Cells (MFC). It also tries to elucidate and effect of mediators on electricity production from wastewaters through experimentation using a simulated wastewater. In order to offset the very high cost of proton exchange membrane, salt bridges have been used in dual chamber MFCs. When the concentration of salt was varied in agar salt bridges from 1% to 10%, the volumetric power density changed from 1.71 to 84.99 mW/m3 with a concomitant variation in power density from 0.32 to 16.02 mW/m2. The maximum power density was observed at 5% salt concentration with 10% agar, which was accompanied by 88.41% COD reduction. In the case of methylene blue (0.01 mM) as the electron mediator, the voltage and current generation were 0.551 V and 0.47 mA, respectively. A maximum open circuit voltage of 0.718 V was seen at 0.08 mM methylene blue concentration, whereas maximum power densities of 17.59 mW/m2 and 89.22 mW/m3 were obtained. Different concentrations of neutral red were also tried out as mediators. A maximum open circuit voltage of 0.730 V was seen at 0.01mM neutral red, corresponding to a power density of 12.02 mW/m2 (volumetric power density of 60.97 mW/m3). Biofilm formation on the electrode surface was not observed in the presence of mediators, but was present in the absence of mediators. The results clearly demonstrated the feasibility to use agar salt bridge for proton transport and role of mediators in MFCs to generate electricity.

Mechanism of Tumor Necrosis Factor alpha (TNFα) Inhibition of B0AT1 Mediated Glutamine Transport in Rat Intestinal Epithelial Cell (IEC‐6)

BackgroundTNFα inhibits B0AT1‐mediated glutamine (Gln) transport by decreasing the affinity of the transporter for Gln on the brush border membrane (BBM) of IEC‐6 cells. TNFα has been shown to exert its effect on several proteins through different signaling pathways.AimTo identify the specific signaling pathway that TNFα activates to inhibit B0AT1‐mediated Gln transport in IEC‐6 cells.MethodsIEC‐6 cells grown on 6‐well plates were treated with TNFα and/or specific inhibitors: Suramin, RpMBcAMP, Calphostin C, BAPTA, and Genistein for PKG, PKA, PKC, Ca2+ chelator, and ERK/JUNK, respectively for 24 h. B0AT1 function was determined by measuring [3H]‐Gln uptake. Cytosolic Ca2+ was measured using Flu 3AM. RTQ‐PCR and Western blot analyses were performed.ResultsTNFα decreased B0AT1 activity in IEC‐6 cells (7.1±0.1 nmol/mg protein/2 min in control and 2.9±0.1 in TNFα treated cells, n=4, p<0.01). Only Genistein fully blocked TNFα mediated inhibition of B0AT1 activity (6.9±0.1 nmol/mg protein/2 min). Kinetic studies demonstrated that the protective mechanism of TNFα mediated inhibition of B0AT1 by Genistein was due to the restoration of affinity of B0AT1 for Gln. Molecular studies showed that both B0AT1 mRNA and B0AT1 protein levels on BBM remained unaltered in all groups.ConclusionsThese studies revealed that TNFα inhibits B0AT1 through ERK/JUNK pathway most likely by phosphorylation in IEC‐6 cells.

SNAREs in HIV-1 assembly

Viruses have a limited number of genes but a complex life cycle and have evolved to utilize numerous host factors to complete their replication. The assembly and budding process of enveloped viruses utilizes numerous cellular factors to facilitate transport from one membrane bound compartment to the other. The host SNARE proteins are widely involved in late stages of vesicular mediated transport by catalyzing the docking and fusion of apposing membranes in the vesicle and target compartment. By generalized disruption of the SNARE sorting machinery, we recently demonstrated a role for these proteins in HIV-1 assembly by affecting Gag localization to the plasma membrane. Whether the observed phenomenon is specifically due to SNARE disruption or generalized disturbance of the cell sorting machinery and the involvement of specific “v” vs. “t” SNAREs in this phenomenon remains to be elucidated.

Monitoring passive transport of redox mediators across a confluent cell monolayer with single-cell resolution by means of scanning electrochemical microscopy

Scanning electrochemical microscopy (SECM) was used for time-resolved studies of passive transport of redox active substances across cell monolayers with sub-cellular resolution. Platinum disk ultramicroelectrodes (UMEs) with effective electrode radii in the sub μm range were operated in the constant-height mode. Monolayers of normal rat kidney (NRK) or Madin Darby canine kidney strain II (MDCK II) epithelial cells served as model cell lines and were grown to confluence on permeable membrane filters to investigate the transport of redox mediators across epithelial barriers. A novel electrochemical cell was developed ensuring reliable mechanical fixation of the growth substrate and preventing membrane vibrations during the SECM operation. The detection of different redox mediators was enabled by potential switching at the UME. This concept proved to be an attractive alternative to the use of dual-microelectrodes or micropipette-microelectrode combinations. The various cell lines were investigated regarding their barrier functions with respect to different redox mediators. Para- and/or trans-cellular transport mechanisms could be distinguished using either hydrophilic ruthenium hexamine chloride or lipophilic ferrocene methanol as redox mediators.

F-Actin Mediated Chromosome Transport

F-actin meshwork contraction is a novel mechanism of intracellular transport that was recently shown to transport chromosomes during meiosis in starfish oocytes. The starfish model system is well suited to high-resolution fluorescence microscopy imaging. We use a high-throughput volume filtering procedure as well as optical flow calculations and spatio-temporal image correlation spectroscopy (STICS). We determine the meshwork pore sizes, node distributions, and branching angles, and relate these structural features to predictions of the recently-proposed “homogeneous contraction” model of transport. We also perform indirect structure analysis by simulating diffusing particles migrating though the F-actin meshwork to probe local F-actin structures and to investigate how F-actin bundle remodeling or binding to F-actin bundles would affect particle motion. We analyze these results in the context of percolation theory and well-understood diffusion model systems. We furthermore compare the simulated particle trajectories to experimental observations of chromosomes and inert PEG-coated beads diffusing within both active and utrophin-stabilized F-actin meshworks. The experimental data are in good agreement with our simulations, and both suggest that chromosome-sized particles can be transported by the F-actin meshwork as passive cargo undergoing confined diffusion.

The Nucleotide-Binding State of Microtubules Modulates Kinesin Processivity and Tau's Ability to Inhibit Kinesin Mediated Transport

Tau's ability to act as a potent inhibitor of kinesin motility in vitro suggests it may actively participate in the regulation of axonal transport in vivo. However, it remains unclear how kinesin based transport could then proceed effectively in neurons, where tau is expressed at high levels. One potential explanation is that tau, a conformationally dynamic protein, has multiple modes of interaction with the microtubule, not all of which are inhibitory for kinesin motility. Thus, if tau can bind microtubules in distinct conformations or at unique binding sites that no longer inhibit kinesin, transport would proceed unhindered along the axon. Previous studies support the hypothesis that tau has at least two modes of interaction with microtubules, but the mechanisms by which tau adopts these different conformations and their functional consequences have not previously been investigated. In the present study we have used single molecule imaging techniques to demonstrate that tau inhibits kinesin motility in an isoform dependent manner on GDP microtubules stabilized with either paclitaxel or glycerol, but not GMPCPP-stabilized microtubules. Furthermore, the order of tau addition to microtubules before or after polymerization has no effect on tau's ability to modulate kinesin motility regardless of the stabilizing agent used. Finally, the processive run length of kinesin is reduced on GMPCPP microtubules relative to GDP-microtubules. These results shed new light on tau's potential role in the regulation of axonal transport, which is more complex than previously recognized.

Periodontal Disease and Pregnancy Outcomes: Time to Move On?

Maternal periodontal disease is a highly prevalent condition that has been studied extensively in relation to adverse pregnancy outcomes, including preterm delivery, preeclampsia, and low birth weight. Investigators speculate that hematogenous transport of bacteria and/or pro-inflammatory mediators from sites of periodontal infection into the placenta, fetal membranes, and amniotic cavity induces pathological processes that lead to these adverse outcomes. Preliminary observational studies supported this hypothesis, but more recent work by our group and others do not demonstrate an increased risk of adverse pregnancy outcomes among women with periodontal disease, and most randomized trials fail to demonstrate improved perinatal outcomes following treatment of periodontal disease in pregnancy.

AtHKT1;1 Mediates Nernstian Sodium Channel Transport Properties in Arabidopsis Root Stelar Cells

The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na+) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na+ and K+ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K+ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na+ over K+ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na+ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na+ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na+ channel transport properties.

Quercetin Increases Cystic Fibrosis Transmembrane Conductance Regulator–Mediated Chloride Transport and Ciliary Beat Frequency: Therapeutic Implications for Chronic Rhinosinusitis

Increasing epithelial chloride (Cl(-)) secretion in the upper airways represents a putative method for promoting mucociliary clearance through augmentation of airway surface liquid depth. Several naturally occurring flavonoid compounds, including quercetin, have shown the capacity to increase transepithelial Cl(-) transport. Quercetin exhibits well-known antioxidant and anti-inflammatory activity and is now recognized as a potent activator of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel activity in a fashion largely independent of cyclic adenosine monophosphate signaling. The present study investigates whether this compound activates Cl(-) secretion and ciliary beat frequency (CBF) in well-characterized culture models of sinonasal epithelium. Cystic fibrosis and non-cystic fibrosis primary human sinonasal epithelial (HSNE) and murine nasal septal epithelial (MNSE) cultures were studied for transepithelial ion transport in Ussing chambers under voltage clamp conditions and CBF was performed using pharmacologic manipulation. Change in short circuit current (DeltaI(SC), expressed as microamperes per squared centimeter) in response to quercetin were significantly greater than controls in both MNSE (23.23 ± 5.44 versus 2.47 ± 1.62; p < 0.0001) and HSNE (-8.72 ± 1.88 versus -1.88 ± 0.66; p < 0.01) cultures. CBF was significantly increased in quercetin-treated cells (expressed as fold change over baseline) in wild type (1.65 ± 0.13 versus 1.23 ± 0.05 [control]; p < 0.01), but not CFTR(-/-) (1.65 ± 0.29 versus 1.48 ± 0.38; p = 0.23). Quercetin significantly increased transepithelial Cl(-) transport and CBF in MNSE and HSNE cultures. Future studies investigating quercetin as a means to promote mucociliary transport in individuals with rhinosinusitis are warranted.

Aspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4–Mediated Transport: Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport.Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4.Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis.Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production).Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Role of Hepatic Lipase and Endothelial Lipase in High-Density Lipoprotein—Mediated Reverse Cholesterol Transport

Reverse cholesterol transport (RCT) constitutes a key part of the atheroprotective properties of high-density lipoproteins (HDL). Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol levels. Although overexpression of EL decreases overall macrophage-to-feces RCT, knockout of both HL and EL leaves RCT essentially unaffected. With respect to important individual steps of RCT, current data on the role of EL and HL in cholesterol efflux are not conclusive. Both enzymes increase hepatic selective cholesterol uptake; however, this does not translate into altered biliary cholesterol secretion, which is regarded the final step of RCT. Also, the impact of HL and EL on atherosclerosis is not clear cut; rather it depends on respective experimental conditions and chosen models. More mechanistic insights into the diverse biological properties of these enzymes are therefore required to firmly establish EL and HL as targets for the treatment of atherosclerotic cardiovascular disease.

Role of Phospholipid Transfer Protein in High-Density Lipoprotein– Mediated Reverse Cholesterol Transport

Reverse cholesterol transport (RCT) describes the process whereby cholesterol in peripheral tissues is transported to the liver where it is ultimately excreted in the form of bile. Given the atherogenic role of cholesterol accumulation within the vessel intima, removal of cholesterol through RCT is considered an anti-atherogenic process. The major constituents of RCT include cell membrane– bound lipid transporters, plasma lipid acceptors, plasma proteins and enzymes, and lipid receptors of liver cell membrane. One major cholesterol acceptor in RCT is high-density lipoprotein (HDL). Both the characteristics and level of HDL are critical determinants for RCT. It is known that phospholipid transfer protein (PLTP) impacts both HDL cholesterol level and biological quality of the HDL molecule. Recent data suggest that PLTP has a site-specific variation in its function. Moreover, the RCT pathway also has multiple steps both in the peripheral tissues and circulation. Therefore, PLTP may influence the RCT pathway at multiple levels. In this review, we focus on the potential role of PLTP in RCT through its impact on HDL homeostasis. The relationship between PLTP and RCT is expected to be an important area in finding novel therapies for atherosclerosis.

Structural Studies of Mammalian Dynactin CAP-Gly Domain by Solid-State NMR

Microtubules and their associated proteins play essential roles in a broad range of physiological functions, including cell migration, mitosis, polarization, differentiation, and vesicle and organelle transport. Dynactin, an activator and the dominant cofactor of dynein, is a large (1.2 mD) multisubunit complex that bridges vesicles to the MT network and thus is central to dynein mediated retrograde transport. Its central subunit, p150Glued, binds to microtubules via the CAP-Gly domain that is thought to be critical for +MT tip localization. Mutations in the p150Glued subunit of the CAP-Gly domain have been implicated in predominantly in neurological disorders including dSMBA and Perry's Syndrome. Despite its critical role, the mechanism by which the CAP-Gly domain recognizes microtubules remains largely unknown, particularly at the atomic level. Herein, we report our recent progress in structural studies of CAP-Gly domain of dynactin by using solid-state NMR. For tertiary structure calculation, we acquired numerous medium- and long-range distance restraints from the DARR and R211 spectra of the 13C sparsely enriched CAP-Gly.

Characterization of Digoxin Uptake in Sandwich-Cultured Human Hepatocytes

Digoxin is a drug that is commonly used to treat congestive heart failure. Because of digoxin's narrow therapeutic index, patients are susceptible to drug-drug interaction-mediated cardiotoxicity. Digoxin is primarily cleared renally; however, a significant component of clearance is due to multidrug resistance 1-mediated transport into bile. Digoxin is reported to be actively transported into human hepatocytes by the organic anion-transporting polypeptide 1B3 (OATP1B3); however, further characterization has not been fully described. The purpose of this study was to investigate the hepatic uptake mechanisms of [(3)H]digoxin using sandwich-cultured human hepatocytes (SCHH) and transporter-expressing cells. Digoxin uptake in SCHH involves both a saturable (carrier-mediated) process and a passive (nonsaturable) process. At low concentrations, the saturable component exhibited an apparent K(m) of 2.39 μM and a V(max) of 4.49 pmol/(min · mg protein). The calculated passive diffusion clearance was 1.25 μl/(min · mg protein). Uptake of [(3)H]digoxin in SCHH was not inhibited by a variety of substrates or inhibitors for OATP1B1, OATP1B3, OATP2B1, organic anion transporter 2, organic cation transporter 1, and monocarboxylate transporter 8. Cytochalasin B, which inhibits glucose transporters, did not significantly inhibit digoxin uptake, whereas the flavonoids quercetin and rutin inhibited uptake by ∼50%. Nonlabeled digoxin inhibited [(3)H]digoxin uptake by ∼50%. Studies with OATP-transfected human embryonic kidney cells or oocytes showed that digoxin is not a substrate of OATP1B1, OATP2B1, or OATP1B3. In conclusion, the data suggest that digoxin uptake in SCHH involves both saturable and passive processes. The saturable process is mediated by an as yet undetermined digoxin transporter(s).