Hikaru Yabuuchi

Molecular and Functional Identification of Sodium Ion-dependent, High Affinity Human Carnitine Transporter OCTN2*

Primary carnitine deficiency, because of a defect of the tissue plasma membrane carnitine transporters, causes critical symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and assembled a cDNA-encoding OCTN2 as a homologue of the organic cation transporter OCTN1, and then we examined the function of OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide of 557 amino acids with 75.8% similarity to OCTN1. Northern blot analysis showed that OCTN2 is strongly expressed in kidney, skeletal muscle, heart, and placenta in adult humans. When OCTN2 was expressed in HEK293 cells, uptake ofl-[3H]carnitine was strongly enhanced in a sodium-dependent manner with K m value of 4.34 μm, whereas typical substrates for previously known organic cation transporters, tetraethylammonium and guanidine, were not good substitutes. OCTN2-mediatedl-[3H]carnitine transport was inhibited by the d-isomer, acetyl-d,l-carnitine, and γ-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, β-hydroxybutyric acid, γ-aminobutyric acid, lysine, and taurine were not inhibitory. Because the observed tissue distribution of OCTN2 is consistent with the reported distribution of carnitine transport activity and the functional characteristics of OCTN2 coincide with those reported for plasma membrane carnitine transport, we conclude that OCTN2 is a physiologically important, high affinity sodium-carnitine cotransporter in humans.

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis (jvs; ref. 6). Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2 (ref. 9). Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1

The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues.

ABCC13, an unusual truncated ABC transporter, is highly expressed in fetal human liver.

In the present study, we have cloned the cDNA of ABCC13, a novel ABC transporter, from the cDNA library of adult human placenta. The ABCC13 gene spans approximately 70kb on human chromosome 21q11.2 and consists of 14 exons. The open reading frame of the ABCC13 cDNA encodes a peptide consisting of 325 amino acid residues. The amino acid sequence corresponding to putative membrane-spanning domains was remarkably similar to ABCC1, ABCC2, ABCC3, and ABCC6. The ABCC13 gene was expressed in the fetal liver at the highest level among the organs studied. While ABCC13 was expressed in the bone marrow, its expression in peripheral blood leukocytes of adult humans was much lower and no detectable levels were observed in differentiated hematopoietic cells. The expression of ABCC13 in K562 cells decreased during cell differentiation induced by TPA. These results suggest that the expression of human ABCC13 is related with hematopoiesis.

Involvement of Multidrug Resistance-Associated Protein 2 (Abcc2) in Molecular Weight-Dependent Biliary Excretion of β-Lactam Antibiotics

In the present study, we attempted to identify the membrane permeation process(es) primarily involved in the molecular-weight-dependent biliary excretion of β-lactam antibiotics. A search of the literature indicated that the molecular weight threshold operates mainly in the transport process across bile canalicular membranes. We confirmed that biliary clearance of the model biliary-excretion-type cephalosporin cefoperazone was reduced to 10% of the control in Eisai hyperbilirubinemic rats, which are genetically deficient in multidrug resistance-associated protein (Mrp) 2, indicating that Mrp2 plays a major role as an efflux transporter on the canalicular membranes. ATP-dependent uptake of several cephalosporins including cefoperazone, cefbuperazone, cefpiramide, and ceftriaxone, all of which are mainly excreted into bile, was confirmed in membrane vesicles from Sf9 cells transfected with rat Mrp2. Both the inhibitory potency of the cephalosporins for Mrp2-mediated transport and the uptake of cephalosporins by Mrp2-expressing vesicles were molecular weight-dependent, suggesting that Mrp2 is one of the major transporters involved in molecular weight-dependent biliary excretion. An uptake study in membrane vesicles of Sf9 cells transfected with breast cancer resistance protein (Bcrp) revealed that Bcrp accepts cefoperazone, cefbuperazone, cefpiramide, cefotetan, ceftriaxone, cefotiam, cefamandole, and cefazolin as substrates, and Bcrp-mediated transport was also molecular weight-dependent, suggesting that Bcrp also contributes to molecular weight-dependent biliary excretion of β-lactam antibiotics in rats.

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush‐border membrane vesicles.

Intestinal Brush‐border Membrane Transport of Monocarboxylic Acids Mediated by Proton‐coupled Transport and Anion Antiport Mechanisms

Intestinal brush‐border membrane transport of monocarboxylic acids was investigated by using rabbit intestinal brush‐border membrane vesicles (BBMVs) and isolated intestinal tissues mounted on Ussing‐type chambers.

Oseltamivir (Tamiflu) Is a Substrate of Peptide Transporter 1

Oseltamivir, an ester-type prodrug of the neuraminidase inhibitor [3R,4R,5S]-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate phosphate (Ro 64-0802), has been developed for the treatment of A and B strains of the influenza virus but has neuropsychiatric and other side effects. In this study, we characterized the transport across intestinal epithelial cells and the absorption of oseltamivir in rats. Uptake by Caco-2 cells (human carcinoma cell line) and HeLa cells transfected with peptide transporter 1 (HeLa/PEPT1) was time- and temperature-dependent and was inhibited by typical PEPT1 inhibitors such as glycyl-sarcosine (Gly-Sar). The uptake by Caco-2 cells and HeLa/PEPT1 was saturable, with similar Km values. Oseltamivir absorption in adult rats was greatly reduced by simultaneous administration of milk, casein, or Gly-Sar. Furthermore, the plasma and brain concentrations of oseltamivir were higher in fasting than in nonfasting rats after oral administration. These results suggest that oseltamivir is a substrate of PEPT1 and that PEPT1 is involved in its intestinal absorption.

Effect of P-glycoprotein on intestinal absorption and brain penetration of antiallergic agent bepotastine besilate

The antiallergic agent bepotastine besilate is a nonsedating, second-generation H1-antagonist with high oral absorption and negligible distribution into brain. To clarify the role of P-glycoprotein (P-gp) in the pharmacokinetics of bepotastine, intestinal absorption and brain penetration studies were performed. [14C]Bepotastine transport in P-gp-overexpressed LLC-PK1 cells indicated that bepotastine was a substrate of P-gp. The affinity of bepotastine to P-gp estimated by ATPase activity assay was low, with a Km value of 1.25 mM. After i.v. administration, the brain/plasma free concentration ratio in mdr1-knockout mice was 3 times higher than that in wild-type mice. The in situ intestinal absorption studies of [14C]bepotastine in rats showed a clear regional difference, showing highest permeability at the upper part of small intestine with a decreasing permeability in the descending part of small intestine. The apparent absorption rate constant (ka) of [14C]bepotastine in the small intestine was greatly increased by cyclosporin A and verapamil, especially in the distal portion, and the site-specific absorption of [14C]bepotastine disappeared. The concentration dependence of ka of [14C]bepotastine was observed with a higher ka at higher concentration (20 mM) compared with that at lower concentration (1 μM). In conclusion, bepotastine is a substrate for P-gp, and P-gp clearly limited the brain distribution of bepotastine, whereas the effect of P-gp on intestinal absorption of bepotastine was minimal, presumably because of high membrane permeability at the upper region of small intestine where P-gp is less expressed. Such intestinal absorption property of bepotastine is distinctly different from the low membrane-permeable P-gp substrate fexofenadine.

Faropenem Transport across the Renal Epithelial Luminal Membrane via Inorganic Phosphate Transporter Npt1

ABSTRACT We previously showed that the mouse inorganic phosphate transporter Npt1 operates in the hepatic sinusoidal membrane transport of anionic drugs such as benzylpenicillin and mevalonic acid. In the present study, the mechanism of renal secretion of penem antibiotics was examined by using a Xenopus oocyte expression system. Faropenem (an oral penem antibiotic) was transported via Npt1 with a Michaelis-Menten constant of 0.77 ± 0.34 mM in a sodium-independent but chloride ion-sensitive manner. When the concentration of chloride ions was increased, the transport activity of faropenem by Npt1 was decreased. Since the concentration gradient of chloride ions is in the lumen-to-intracellular direction, faropenem is expected to be transported from inside proximal tubular cells to the lumen. So, we tested the release of faropenem from Xenopusoocytes. The rate of efflux of faropenem from Npt1-expressing oocytes was about 9.5 times faster than that from control water-injectedXenopus oocytes. Faropenem transport by Npt1 was significantly inhibited by β-lactam antibiotics such as benzylpenicillin, ampicillin, cephalexin, and cefazolin to 24.9, 40.5, 54.4, and 26.2% of that for the control, respectively. Zwitterionic β-lactam antibiotics showed lesser inhibitory effects on faropenem uptake than anionic derivatives, indicating that Npt1 preferentially transports anionic compounds. Other anionic compounds, such as indomethacin and furosemide, and the anion transport inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid significantly inhibited faropenem uptake mediated by Npt1. In conclusion, our results suggest that Npt1 participates in the renal secretion of penem antibiotics.