Masayuki Ohbayashi

Transport mechanism and substrate specificity of human organic anion transporter 2 (hOat2 [SLC22A7])

Human organic anion transporter 2 (hOat2 [SLC22A7]) is highly expressed in the human liver. Although localization, gene expression, substrate specificity and transport mechanisms of other human Oat isoforms such as human Oat1 (hOat1), human Oat3 (hOat3) and human Oat4 (hOat4) have been elucidated, information concerning human Oat2 (hOat2) is less defined. The objective of this study was to provide further information on the transport mechanism and substrate specificity of hOat2. When expressed in Xenopus laevis oocytes, the transport of organic compounds mediated by hOat2 was not affected by the replacement of extracellular sodium with lithium, choline and mannitol. The uptake of estrone sulfate (ES) in hOat2‐expressing oocytes was significantly trans‐stimulated by preloading the oocytes with fumarate and succinate, but not glutarate. Moreover, we observed that hOat2 mediates the transport of bumetanide, ES, glutarate, dehydroepiandrosterone sulfate, allopurinol, prostaglandin E2, 5‐fluorouracil, paclitaxel and L‐ascorbic acid. These compounds are identified for the first time as hOat2 substrates. A wide range of structurally unrelated organic compounds inhibited the hOat2‐mediated uptake of tetracycline, except for sulfobromophthalein. All of these findings indicate that hOat2 is a sodium‐independent multi‐specific organic anion/dimethyldicarboxylate exchanger. Our present findings thus provide further insights into the role of hOat2 in hepatic drug transport.

FUNCTIONAL CHARACTERIZATION OF HUMAN MONOCARBOXYLATE TRANSPORTER 6 (SLC16A5)

Human monocarboxylate transporter 6 (MCT6) has recently been isolated, and its tissue distribution has been established at the mRNA level, but its functional properties remain unknown. The aim of this study is to investigate the transport properties of MCT6. When expressed in Xenopus laevis oocytes, MCT6 transported [3H]bumetanide in a pH- and membrane potential-sensitive but not proton gradient-dependent manner, with the Kt value of 84 μM. Furthermore, MCT6 transported various drugs such as probenecid and nateglinide. Neither [14C]l-lactic acid nor [3H]l-tryptophan, typical substrates of other MCT isoforms, was transported by MCT6. Four loop diuretics, i.e., furosemide, piretanide, azosemide, and torasemide, thiazides, probenecid, glibenclamide, and nateglinide inhibited the MCT6-mediated uptake of [3H]bumetanide. In contrast, short-chain carboxylic acids, such as l-lactic acid and succinic acid did not inhibit the MCT6-mediated uptake of bumetanide. These results suggest that the substrate specificity of MCT6 is distinct from those of other MCTs. Bumetanide would be a good tool for investigating the functional properties of MCT6. It is probable that MCT6 is involved in the disposition of various drugs, including bumetanide.

Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1).

We succeeded in isolating the cDNA-encoding mouse organic solute carrier protein 1 (mOscp1) from a mouse testis cDNA library. mOscp1 consisted of 1137 base pairs that encoded a 379-amino acid protein, and the amino acid sequence was 85% identical to that of human OSCP1 (hOSCP1). Northern blot analysis revealed that the gene coding for mOscp1 is highly expressed in the testis, but not in other tissues. When expressed in Xenopus laevis oocytes, mOscp1 mediated the high-affinity transport of p-aminohippurate (PAH) (Km = 18.8 ± 4.1 μM) with Na+ independence. mOscp1 transported various kinds of structurally dissimilar drugs and chemicals such as probenecid, dehydroepiandrosterone sulfate, and glutarate with some differences in substrate specificity compared with hOSCP1. Cyclophosphamide inhibited the mOscp1-mediated PAH uptake. Immunohistochemical analysis revealed that the mOscp1 protein is localized in the plasma membrane side of Sertoli cells in the testis. Our results indicate that isolated mOscp1 is a polyspecific organic solute carrier protein and may be a key molecule for the testicular handling of organic solutes.

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation.

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca(2+)-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Characterization of monocarboxylate transporter 6: expression in human intestine and transport of the antidiabetic drug nateglinide

Monocarboxylate transporter (MCT) 6, encoded by SLC16A5, is a member of the monocarboxylate transporter family. Nateglinide, an oral hypoglycemic agent, quickly reaches the maximal serum concentration after its premeal administration. Although the functional existence of uptake systems for nateglinide in the intestine has been demonstrated, these transport systems have not yet been identified at the molecular level. The aim of this study was to demonstrate the localization of MCT6 in the human small intestine and characterize the transport properties of nateglinide via MCT6. Immunohistochemical analysis of the human small intestine revealed that anti-MCT6 antiserum stained the luminal side of the epithelial cells. When expressed in Xenopus laevis oocytes, MCT6-mediated uptake of [14C]nateglinide was sensitive to extracellular pH and membrane potential. Furthermore, the Kt value of nateglinide (45.9 μM) for MCT6 was lower than those previously reported in Caco-2 cells and rat intestinal brush-border membrane vesicles. In addition, probenecid, fluorescein, valproic acid, and salicylic acid, which are inhibitors of nateglinide uptake in Caco-2 cells and rat intestine, did not inhibit the uptake of nateglinide via MCT6. These results suggest that MCT6 may play a role in the intestinal absorption of nateglinide, although other transporters are also likely involved.

Genetic polymorphism of the human organic solute carrier protein 1 (hOSCP1) gene in Japanese patients with non-viral liver carcinoma.

Human organic solute carrier protein 1 (hOSCP1) is a Na+-independent multispecific organic solute transporter. To date, several studies have revealed that gene mutations of the transporters are likely to be associated with some diseases; however, there are no data concerning the genetic polymorphism of the hOSCP1 gene in Japanese patients with non-viral liver carcinoma (LC). In the present study, we isolated genomic DNA from a normal portion of LC, and analyzed 41 single nucleotide polymorphisms (SNPs) chosen from a database of SNPs (dbSNPs). We found genotype frequencies for 2 non-synonymous SNPs [rs34409118 (Thr131 → Ala) and rs1416840 (Ile219 → Thr)] and 1 synonymous SNP [rs16822954 (Ser193 → Ser)] to be statistically significant when compared with dbSNPs. No statistical significance was observed in rs2275477 (Gly307 → Arg) in the hOSCP1 gene. With respect to the allele frequency, we also observed rs34409118 to be statistically significant. Interestingly, we found that non-viral LC patients do not carry heterozygous mutations in rs1416840 (A/G) and rs16822954 (A/G), suggesting that a non-carrier of heterozygous mutations in these two SNPs might be a biomarker for susceptibility for non-viral LC in Japanese. Further analyses of patients with hOSCP1 variants may elucidate the relationship between the hOSCP1 gene and susceptibility of non-viral LC in Japanese patients.

Activation of Cyclosporin A Transport by a Novel λ Light Chain of Human Ig Surface Antigen-Related Gene in Xenopus laevis Oocytes

In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the λ light chain of human Ig surface antigen-related gene (IgLC-rG). The isolated cDNA consisted of 693 base pairs that encoded a 232-amino acid protein. Northern blot analysis revealed that the IgLC-rG mRNA is expressed in the adult spleen and small intestine but not in fetal tissues. When expressed in Xenopus laevis oocytes, IgLC-rG mediated the high-affinity transport of [3H]cyclosporin A (CsA) (Km = 189.7 ± 123.5 nM) in a sodium-dependent manner; however, other organic solutes such as p-aminohippuric acid and TEA were not transported via IgLC-rG. The transport of [3H]CsA by IgLC-rG was sensitive to pH. The uptake of [3H]CsA was trans-stimulated by CsA and GSH. Immunohistochemical analysis revealed that the IgLC-rG protein is localized at the brush border membrane in the human small intestine. Although the isolated IgLC-rG gene is a member of the human Ig λ light chain surface antigen superfamily, our findings suggest that IgLC-rG functions as a novel transport peptide responsible for CsA in the human body. Our results should provide insight into the novel function of membrane-bound proteins, such as Igs.

Effects of riluzole on spinal seizure-like activity in the brainstem-spinal cord preparation of newborn rat

Riluzole blocks persistent Na+ current, inhibits generation of neuronal bursts and decreases glutamate-induced excitotoxicity. In previous studies of respiratory activity, riluzole suppressed inspiratory-related burst generation activity in rat slice or en bloc preparations. We examined riluzoles effects on inspiratory burst generation and drug-induced seizure-like activity in newborn rat en bloc preparations. Medulla-spinal cord preparations from postnatal day 0-3 Wistar rats were isolated under deep isoflurane anesthesia and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25-26°C. Inspiratory activity was monitored from the fourth cervical ventral root. Seizure-like activity was induced by application of 20μM DL-threo-β-benzyloxyasparatate (TBOA, a glutamate uptake blocker preferentially acting on astrocytes) or coadministration of GABAA antagonist bicuculline (10μM) and glycine antagonist strychnine (10μM). Pretreatment and co-application with 10μM riluzole abolished the seizure-like burst activity induced by TBOA or bicuculline/strychnine. N-methyl-d-aspartic acid receptor antagonist MK801 (10μM) also depressed this activity. Riluzole may attenuate excessive glutamate action involved in pathological hyperexcitability of motor neurons with no major effect on generation of respiratory activity. Riluzole at the optimal dose could be a potential treatment to protect drug-induced epileptic brain tissue from excitotoxic damage without inducing respiratory suppression.

Angiogenesis induced by adhesion between polymorphonuclear leukocyte and endothelial cell via intercellular adhesion molecule-1

We demonstrate that adhesion between polymorphonuclear leukocyte (PMN) and endothelial cells (ECs) is concerned with induction of angiogenesis of ECs. For the tube formation assay, ECs obtaine from bovine thoracic aorta (BAECs) grown on a layer of collagen type I were used. Addition of PMNs treated with N-formyl-methionyl-leucy phenylalanine (FMLP), a selective activator of PMN induced angiogenesis The angiogenesis was blocked by monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1) which inhibit the adhesion between PMN and EC. Ets-1, which stimulates metalloproteinase gene transcription or integrin s3, has a key role in angiogenesis. Addition of activate PMNs to ECs stimulated the angiogenesis and Ets-1 expression. Both the angiogenesis and the Ets-1 expression induced by PMNs were reduced by ets-1 antisense oligonucleotide. On the other hand, PMN-induced Ets-1 expression was reduced by a monoclonal antibody against ICAM-1 but not E-selectin despite the inhibition of PMN-induced angiogenesis by both antibodies. The enhancement of angiogenesis by FMLP-treated PMNs Was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). Interestingly, the stimulation of angiogenesis by H2O2 without PMNs was inhibited by anti E-selectin antibody but not anti ICA1V 1. ICAM-1 stimulation occurred by ICAM-1 cross-linking enhanced angio genesis. These findings indicated that PMN adhesion was related with the induction of angiogenesis, and ICAM-1 in endothelial cells acted as a signaing receptor to induce Ets-1 expression, whereas E-selectin seemed to function in the formation of tube-like structures in vascular endothelial cell cultures.