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H Endou

Publications and source records attributed to H Endou.

At least 55 records · Page 3Linked to original sources
PubMed2000

Purinoceptor-mediated calcium mobilization and cellular proliferation in cultured bovine corneal endothelial cells.

In the present study, we investigated the effect of adenosine triphosphate (ATP) on cytosolic free calcium mobilization and mitogenic activity in cultured bovine corneal endothelial cells (BCEC). The [Ca2+]i was determined using a Ca2+ sensitive indicator, Fura-2/AM, and cell proliferation was evaluated by counting the cell number. ATP, its metabolites and analogs caused transient increase in [Ca2+]i in a concentration-dependent manner (10(-7) M-10(-3) M) and the potency of agonists was ordered as follows: 2-methylthio-ATP > uridine triphosphate > ATP > adenosine diphosphate. Adenosine monophosphate and adenosine did not affect [Ca2+]i. ATP (10(-4) M) also promoted the accumulation of inositol trisphosphate (IP3). The ATP-induced transient [Ca2+]i increase and IP3 accumulation were attenuated by pretreatment with a phospholipase C inhibitor, U-73122 (5 microM), for 30 min. ATP (10(-5) M) significantly enhanced the proliferation of BCEC. ATP-induced [Ca2+]i increase and cell proliferation were inhibited by a purinoceptor antagonist, suramin (10(-4) M). Thus, the present study indicates that BCEC contain P2 purinoceptors that regulate their proliferation.

Adenosine Triphosphate↗
PubMed2000

Rat multispecific organic anion transporter 1 (rOAT1) transports zidovudine, acyclovir, and other antiviral nucleoside analogs.

Organic anion transporter 1 (OAT1) is a p-aminohippurate/dicarboxylate exchanger that plays a primary role in the tubular secretion of endogenous and exogenous organic anions. OAT1 is located in the basolateral membrane of the proximal tubular cells and mediates the uptake of various organic anions from the peritubular fluid. In this study, we investigated the transport of antiviral nucleoside analogs via rat OAT1 (rOAT1) using a heterologous expression system in Xenopus laevis oocytes. Oocytes injected with rOAT1 cRNA showed significantly higher uptake of zidovudine (AZT) and acyclovir (ACV) than control oocytes. rOAT1-mediated uptake of AZT and ACV was probenecid-sensitive and increased by the outwardly directed gradient of glutarate. The affinity of rOAT1 for AZT and ACV was determined to be 68 and 242 microM, respectively. Five other antiviral agents that we studied (zalcitabine, didanosine, lamivudine, stavudine, and trifluridine) were also shown to be transported by rOAT1, whereas foscarnet, a phosphate analog, was not. The aforementioned nucleoside analogs lack a typical anionic group and are not very hydrophobic. This study demonstrates extension of the substrate spectrum of rOAT1 and provides a molecular basis for the pharmacokinetics of antiviral nucleoside analogs.

Acyclovir↗
PubMed2000

[Molecular mechanisms of drug transport].

Drugs ingested into the body are transported through the plasma membrane several times. Although hydrophobic low molecules may penetrate the cell membrane according to simple diffusion, most drugs need carrier proteins named transporters for their trans-membrane transport. Drug transporters are classified into 5 groups by their difference in molecular structures, substrate specificities and transport mechanisms. The five groups of drug transports are as follows: (1) organic ion transporter superfamily, (2) ATP-dependent transporter superfamily, (3) peptide transporter family, (4) organic anion transporting polypeptide family originated from liver and (5) amino acid-polyamine-choline transporter superfamily. Among the 5 groups, there are seen several common characteristics such as structures having 12-transmembrane spanning domains, multispecificity in substrate recognition, different tissue distribution and functional regulation by phosphorylation. Multiple substrate specificities of drug transporters are tentatively understood by their evolution, in which at earlier stages these molecules might be essential to absorb endogenously necessary nutrients, and thereafter structural homologs like drugs might be recognized by these transporters. Several topics remain to be elucidated such as functional significance of drug transporters in pharmacokinetics including drug/drug interaction, polymorphism of drug transporter genes, and molecular mechanisms of substrate recognition by drug transporters.

Biological Transport↗
PubMed1999

Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein.

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b(0,+) amino acid transporter). The transporter designated BAT1 (b(0, +)-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y(+)L, and system x(-)C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na(+)-independent transport of cystine as well as basic and neutral amino acids with the properties of system b(0,+). The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.

ATP-Binding Cassette Transporters↗
PubMed1999

Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain.

A cDNA encoding the new member of the multispecific organic anion transporter family, OAT3, was isolated by the reverse transcription-polymerase chain reaction cloning method. Degenerate primers were designed based on the sequences conserved among OAT1, OAT2, and organic cation transporter 1 (OCT1), and reverse transcription-polymerase chain reaction was performed using rat brain poly(A)+ RNA. The 536-amino acid protein sequence encoded by OAT3 showed 49, 39, and 36% identity to those of OAT1, OAT2, and OCT1, respectively. Northern blot analysis revealed that rat OAT3 mRNA is expressed in the liver, brain, kidney, and eye. When expressed in Xenopus laevis oocytes, OAT3 mediated the uptake of organic anions, such as p-aminohippurate (Km = 65 microM), ochratoxin A (Km = 0.74 microM), and estrone sulfate (Km = 2.3 microM) and a cationic compound, cimetidine. OAT3-mediated uptake of [3H]estrone sulfate was sodium-independent. para-Aminohippuric acid, estrone sulfate or ochratoxin A did not show any trans-stimulatory effect on either influx or efflux of [3H]estrone sulfate via OAT3. Organic anions such as sulfobromophthalein, probenecid, indocyanine green, bumetanide, piroxicam, furosemide, azidodeoxythymidine, 4, 4'-diisothiocyanostilbene-3,3'-disulfonic acid, and benzylpenicillin inhibited OAT3-mediated estrone sulfate uptake, while ouabain and digoxin did not. Organic cations such as tetraethylammonium, guanidine, verapamil, and quinidine did not interact with OAT3. Acidic metabolites of neurotransmitters derived from dopamine, epinephrine, norepinephrine, and serotonin inhibited the uptake of estrone sulfate via OAT3. These results suggest an important role of OAT3 in the excretion/detoxification of endogenous and exogenous organic anions, especially from the brain.

Amino Acid Sequence↗
PubMed1999

Effect of chronic K+ deficiency on contractile properties of soleus muscle in rats: evidence of sex differences.

1. Alterations in skeletal muscle function of chronically K(+)-depleted male and female rats were investigated in isolated soleus muscles. 2. By 16 weeks K+ deficiency, plasma K+ concentrations in both male and female rats were reduced to approximately 2 mEq/L, which was accompanied by an approximate 50% reduction in muscle K+ content and a marked increase in muscle Na+ content. These changes were similar in both males and females. 3. Plasma creatine phosphokinase activity progressively increased with time in K(+)-depleted male rats, whereas only a slight increase was observed in female rats. 4. Maximum isometric twitch tension (Pt) and tetanic tension (Po) of K(+)-depleted soleus muscles from male rats was markedly suppressed; this was not seen for soleus muscles obtained from female rats. 5. After exposure to insulin in low-K+ solution, the contractile tension of soleus from the K(+)-depleted male rats was suppressed to a greater extent. 6. All alterations in muscle function during chronic K+ depletion were restored to normal after 2 weeks K+ repletion. 7. The results suggest that there is a preponderance for male over female rats in developing alterations in skeletal muscle function during chronic K+ deficiency. The changes may be associated with abnormalities of muscle membrane permeability and cellular function.

Animals↗
PubMed1999

Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity.

UNLABELLED: Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity. BACKGROUND: Cephaloridine (CER) has been used to elucidate the mechanisms of cephalosporin antibiotic-induced nephrotoxicity. Organic anion transporters have been thought to mediate CER uptake by the proximal tubule. The purpose of this study was to elucidate the possible involvement of organic anion transporter 1 (OAT1) in CER-induced nephrotoxicity. METHODS: A mouse terminal proximal straight tubule (S3) cell line stably expressing rat OAT1 (S3 rOAT1) was established and used in this study. The cellular uptake of [14C]-para-aminohippuric acid (PAH), a prototype organic anion, and that of [14C]-CER were measured. The effects of CER on the viability of the cells and the amount of lipid peroxidation were estimated. RESULTS: S3 rOAT1 expressed a functional organic anion transporter in the cytoplasmic membrane, and exhibited CER uptake activity. CER treatment resulted in a more significant decrease in the viability and a more significant increase in the amount of lipid peroxidation in S3 rOAT1 than in S3 cells transfected with an expression vector lacking the rOAT1 insert. Probenecid, an inhibitor of organic anion transport, and probucol, an antioxidant, significantly suppressed the decrease in viability and increase in the amount of lipid peroxidation in S3 rOAT1 treated with CER. The effects of various cephalosporin antibiotics on the uptake of [14C]PAH were correlated significantly with the effects of these drugs on cell viability. CONCLUSIONS: These results suggest that rOAT1 is, at least in part, responsible for the cellular uptake of CER and therefore CER-induced nephrotoxicity.

Animals↗
PubMed1999

Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney.

Recently, we isolated the multispecific organic anion transporter (OAT1) from the rat kidney, which plays important roles in the renal elimination of endogenous and exogenous organic anions including clinically important drugs. In the present study, we cloned and characterized human OAT1. Two cDNA clones, hOAT1-1 cDNA and hOAT1-2 cDNA, were isolated from a human kidney cDNA library, whose amino acid sequences were 86.0% and 87.8% identical to that of rat OAT1, respectively. When expressed in Xenopus laevis oocytes, hOAT1 mediated sodium-independent uptake of p-aminohippurate (PAH) (Km = 9. 3 +/- 1.0 microM). hOAT1-mediated PAH uptake was inhibited by bulky inorganic anions, various xenobiotics, and endogenous substances, including benzylpenicillin, furosemide, indomethacin, probenecid, phenol red, urate, and alpha-ketoglutarate. Northern blot analysis revealed that hOAT1 mRNA is strongly expressed in human kidney; transcripts of different sizes are expressed in skeletal muscle, brain, and placenta. Immunohistochemical analysis using rabbit IgG antibody against the carboxy-terminal 14 peptides of hOAT1 revealed that hOAT1 is expressed at the basolateral membrane of the proximal tubule. hOAT1 gene was located on human chromosome 11q13.1 by fluorescent in situ hybridization analysis. These results indicate that hOAT1 is a multispecific organic anion transporter on the basolateral membrane of the proximal tubule in human kidney.

Amino Acid Sequence↗
PubMed1999

Hydrogen peroxide induces necrosis, apoptosis, oncosis and apoptotic oncosis of mouse terminal proximal straight tubule cells.

Hydrogen peroxide (H2O2) has been shown to be an important mediator of ischemic and toxic tubular damage. The purpose of this study was to identify the mode of cell death observed in H2O2-exposed cultures of mouse terminal proximal straight tubule (S3) cells. H2O2 induced a dose- and time-dependent decrease in viability of S3 cells. Morphologically, S3 cells exposed to H2O2 (0.05-0.1 mM) showed features of necrosis, apoptosis, oncosis and apoptotic oncosis, whereas necrosis occurred most frequently in every experimental condition tested. On the other hand, agarose gel electrophoresis of DNA extracted from S3 cells exposed to H2O2 revealed a typical DNA ladder pattern. These data suggest that H2O2-induced proximal tubule damages are associated with the induction of various modes of cell death including necrosis, apoptosis, oncosis and apoptotic oncosis, and with the activation of endonuclease.

Animals↗
PubMed1999

[Amino acid transporter molecule as a drug target].

Amino acids, especially essential amino acids are required for protein synthesis and as energy sources in all living cells. Since most amino acids are hydrophilic, special membrane proteins are necessary for their transmembrane transport particularly in transformed cells. We cloned a cDNA encoding L-type amino acid transporter, and named it LAT1. LAT1 is highly expressed in most tumor cells and in a few normal tissues such as the bone marrow, spleen, placenta and brain. As a homolog of LAT1, we also cloned LAT2 which is expressed in various normal tissues. In order to clarify the role of LAT1 in tumor cells, we tested a relatively specific inhibitor to LAT1, BCH (2-aminobicyclo-2(2,2,1)-heptane-2-caboxylic acid), and found that BCH inhibited mortality of mice treated with sarcoma cells. In addition, BCH also suppressed strongly T24 carcinoma cell growth. Moreover, LAT1 transports amino acid mimetic drugs. Thus, these results indicate that amino acid transporter molecule such as LAT1 can be a drug target.

Amino Acid Transport Systems↗
PubMed1999

Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney.

Renal proximal convoluted tubules have an important role, i.e., to excrete organic anions, including numerous drugs and endogenous substances. Recently, multispecific organic anion transporter 1 (OAT1) was isolated from rat kidney. In this study, the cellular and subcellular localization of OAT1 in rat kidney was investigated. Kidneys from normal rats were perfused and fixed with periodate-lysine-paraformaldehyde solution and were then processed for immunohistochemical analysis using the labeled streptavidin-biotin method, preembedding horseradish peroxidase method, and immunogold method. Light microscopic examination revealed immunostaining for OAT1 in the middle portion of the proximal tubule (S2 segment), but not in the initial portion of the proximal convoluted tubule, next to the glomerulus. Nephron segments other than the S2 segment and the renal vasculature were not stained with antibody to OAT1. Electron-microscopic observation using a preembedding method revealed that OAT1 was exclusively expressed in the basolateral membrane of S2 segments of proximal tubules. The immunogold method showed no labeling for OAT1 in the cytoplasmic vesicles, suggesting that OAT1 may not move together with organic anions into the cells. These results are consistent with previous physiologic data showing that organic anions, including para-aminohippurate, are taken up by the basolateral Na+-independent organic anion/dicarboxylate exchanger and excreted at S2 segments. In conclusion, OAT1 was localized to the basolateral membrane of S2 segments of proximal tubules in rat kidneys.

Animals↗
PubMed1999

T-1095, an inhibitor of renal Na+-glucose cotransporters, may provide a novel approach to treating diabetes.

T-1095A and T-1095 are synthetic agents derived from phlorizin, a specific inhibitor of Na+-glucose cotransporters (SGLTs). Unlike phlorizin, T-1095 is absorbed into the circulation via oral administration, is metabolized to the active form, T-1095A, and suppresses the activity of SGLTs in the kidney. Orally administered T-1095 increases urinary glucose excretion in diabetic animals, thereby decreasing blood glucose levels. Indeed, the postprandial hyperglycemia after a meal load was shown to be suppressed by this compound in streptozotocin (STZ)-induced diabetic rats. With long-term T-1095 treatment, both blood glucose and HbA1c levels were reduced in STZ-induced diabetic rats and yellow KK mice. In addition, there was amelioration of abnormal carbohydrate metabolism, i.e., hyperinsulinemia and hypertriglyceridemia, and of the development of microalbuminuria, in yellow KK mice. Thus, T-1095 may be a useful antidiabetic drug, providing a novel therapeutic approach for diabetes.

Administration, Oral↗
PubMed1999

Silica increases cytosolic calcium and causes cell injury in renal cell lines.

The purpose of this study was to clarify the effect of silica-induced cytosolic free calcium mobilization and cell injury in immortalized cell lines from transgenic mice kidney harboring SV40 T-antigen gene. The proximal convoluted tubule (S1)- and the inner medullary collecting tubule (IMCT)-originated cell lines were used. Cytosolic free calcium concentration ([Ca2+]i) was measured employing Fura-2 fluorescence and cell injury was evaluated by a vital dye exclusion procedure. Silica increased [Ca2+]i in a concentration-dependent manner in S1 (60 micrograms/ml-600 micrograms/ml) and IMCT (6 micrograms/ml-600 micrograms/ml). Silica caused a biphasic increase in [Ca2+]i which was composed of an initial rapid rise and following sustained phase. Ca2+ removal from the medium resulted in abolishment of initial and sustained phase of silica (600 micrograms/ml)-induced [Ca2+]i in both cell lines. Silica-induced cell injury was increased in a dose-dependent manner. This silica-induced cell injury was attenuated by the pretreatment with EGTA (100 microM) and nifedipine (1 microM). Cellular ATP content ([ATP]i) by silica also decreased in a concentration-dependent manner. The relationship between [Ca2+]i and [ATP]i showed that [ATP]i depletion caused [Ca2+]i to rise. This study suggests that 1) the elevation of [Ca2+]i caused by silica is due mainly to influx through plasma membrane Ca2+ channel and non specific membrane damage (at high concentration) and 2) nephrotoxicity of silica shows site-specificity within the kidney.

Adenosine Triphosphate↗
PubMed1999

Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis oocytes.

Organic anion transporter 1 (OAT1) is the para-aminohippurate (PAH) transporter in the basolateral membrane of the proximal tubule. The present study investigated whether or not nonsteroidal anti-inflammatory drugs (NSAIDs) are transported by OAT1. All of the NSAIDs tested inhibited [14C]PAH uptake via OAT1 expressed in Xenopus laevis oocytes. Ibuprofen, indomethacin, salicylurate, and naproxen showed the strongest potency to inhibit [14C]PAH uptake (Ki approximately 2-10 microM); acetylsalicylate, salicylate, and phenacetin exhibited moderate potency (Ki approximately 300-400 microM), and acetaminophen (paracetamol) exhibited the weakest inhibitory potency (Ki approximately 2 mM). Radiolabeled acetylsalicylate, salicylate, and indomethacin were taken up by OAT1 and the uptake rate of these three NSAIDs was enhanced by the outwardly directed dicarboxylate gradient. The efflux of the preloaded [14C]PAH from the oocytes via OAT1 was trans-stimulated by PAH and glutarate added to the media. The addition of salicylate, acetylsalicylate, or salicylurate into the media also trans-stimulated the efflux of PAH, whereas indomethacin did not. The present study indicates that OAT1 is responsible for the renal uptake and secretion of NSAIDs.

Animals↗
PubMed1999

Transport of ochratoxin A by renal multispecific organic anion transporter 1.

In the present study, we investigated the transport of ochratoxin A (OTA) by kidney-specific organic anion transporter 1 (OAT1). When expressed in Xenopus laevis oocytes, OAT1 mediated sodium-independent uptake of OTA (Km = 2.1 microM). Piroxicam, which has been shown to prevent the nephrotoxicity of OTA, inhibited OAT1-mediated uptake of OTA. By contrast, another protective compound, aspartame, did not. Using a cell line derived from the mouse kidney terminal proximal tubule (S3) transfected with OAT1 cDNA, we investigated the transport of OTA and also its effect on cell proliferation and cell viability. S3 cells expressing OAT1 mediated the saturable transport of OTA (Km = 0.57 microM). Cell proliferation was suppressed in S3 cells expressing OAT1 when exposed to 2 and 10 microM OTA. This suppression was rescued by the coaddition of 1 mM p-aminohippurate in the media. The present study indicates that OTA is transported by OAT1 and that the accumulation of OTA via OAT1 in proximal tubular cells is the primary event in the development of OTA nephrotoxicity.

Animals↗
PubMed1999

The interaction and transport of beta-lactam antibiotics with the cloned rat renal organic anion transporter 1.

In the present study, we investigated the interactions between antibiotics, especially beta-lactam antibiotics, and rat renal organic anion transporter 1 (OAT1). [(14)C]p-Aminohippurate (PAH) uptake via OAT1 expressed in Xenopus laevis oocytes was inhibited by all of the penicillins and cephalosporins tested. Penicillin G, carbenicillin, cephaloridine, cephalothin, cefazolin, and cephalexin inhibited [(14)C]PAH uptake via OAT1 in a competitive manner (K(i) = 0.29-2.33 mM). Cinoxacin, a quinolone gyrase inhibitor, also inhibited PAH uptake via OAT1. Other antibiotics, such as gentamicin, streptomycin, and vancomycin, which do not contain anionic moieties, did not interact with OAT1. [(3)H]Penicillin G and [(14)C]cephaloridine were demonstrated to be transported via OAT1. Using the cells that stably expressed OAT1, we analyzed the cytotoxicity of several beta-lactam antibiotics. Cells expressing OAT1 showed higher susceptibility to cephaloridine (a potentially nephrotoxic beta-lactam antibiotic) toxicity than did control cells. The present study suggests that OAT1 is the major organic anion transporter in the kidney that is responsible for the renal secretion of antibiotics, especially that of beta-lactam antibiotics. Furthermore, the culture cell system expressing OAT1 was revealed to be useful for the prediction of the nephrotoxicity of beta-lactam antibiotics.

Animals↗