Biomedical subjects

Masahiro Okuda

Publications and source records attributed to Masahiro Okuda.

At least 19 recordsLinked to original sources

PubMed2006

Androgen receptor is responsible for rat organic cation transporter 2 gene regulation but not for rOCT1 and rOCT3.

PURPOSE: Organic cation transporters 1-3 (OCT1-3; Slc22a1-3) mediate the membrane transport of organic cations in the kidney. We previously reported that rat (r)OCT2 expression in the kidney was regulated by testosterone. In this study, we examined the transcriptional mechanisms underlying the testosterone-dependent regulation of rOCT2 expression. METHODS: Approximately 3000-bp fragments of the rOCT1-3 promoter region were isolated, and promoter activities were measured in the renal epithelial cell line LLC-PK1 with the coexpression of rat androgen receptor. RESULTS: Among reporter constructs tested, only rOCT2 promoter activity was stimulated by testosterone. This stimulation was suppressed by nilutamide, an antiandrogen drug. Reporter assays using deletion constructs and mutational constructs of putative androgen response elements (ARE) in the rOCT2 promoter region suggested that two AREs, located at approximately -3000 and -1300, respectively, play an important role in the induction by testosterone. CONCLUSIONS: Testosterone induces the expression of rOCT2, but not of rOCT1 and rOCT3, via the AR-mediated transcriptional pathway. This is the first study to address the transcriptional mechanisms of testosterone-dependent gene regulation of the Slc22 family.

Androgens↗

PubMed2006

Clinical evaluation of plasma free phenytoin measurement and factors influencing its protein binding.

The relationship between free phenytoin concentrations and clinical responses, and the factors influencing protein binding of phenytoin were investigated. A total of 119 plasma samples from 70 patients treated orally with phenytoin were analysed. The mean free phenytoin concentration was significantly higher in the patients who received phenytoin monotherapy and were classified as having a complete response (1.25 +/- 1.09 microg/ml) than that in the partial response group (0.59 +/- 0.07 microg/ml), whereas the mean total concentrations were not significantly different between the two groups. Samples were divided into three groups based on the free fraction of phenytoin, i.e. low, <5%; medium, 5%-10%; high, > 10%. The mean age (55.3 +/- 10.9 years) was significantly higher in the high group than in the low (42.7 +/- 21.2 years) and medium (42.8 +/- 16.0 years) groups. The mean creatinine clearance (CLcr) (55.3 +/- 10.9 ml/min) and serum albumin concentration (3.30 +/- 1.25 g/dl) were significantly lower in the high group than the low (88.3 +/- 29.0 ml/min and 4.08 +/- 0.50 g/dl, respectively) and medium (95.0 +/- 32.8 ml/min and 3.95 +/- 0.92 g/dl, respectively) groups. These results suggest that the free phenytoin concentration, rather than the total concentration, is more useful for monitoring antiepileptic effects in patients receiving phenytoin monotherapy. It was also found that the free phenytoin fraction was significantly influenced by aging, CLcr and serum albumin levels.

Adolescent↗

PubMed2006

Interactions of fluoroquinolone antibacterials, DX-619 and levofloxacin, with creatinine transport by renal organic cation transporter hOCT2.

Interactions of DX-619, a novel fluoroquinolone antibacterial, and levofloxacin (LVFX) with the human renal organic cation transporter hOCT2 were studied. The intracellular accumulation of [(14)C]creatinine in stable transfectants of HEK293 cells expressing hOCT2 (hOCT2-HEK293) as well as vector-transfected HEK293 cells (VEC-HEK293) was evaluated in the presence of DX-619 and LVFX at various concentrations. When added extracellularly, both DX-619 and LVFX inhibited the uptake of [(14)C]creatinine (5 microM) by hOCT2-HEK293 cells in a dose-dependent manner. Unlike in hOCT2-HEK293 cells, the uptake in VEC-HEK293 cells was not inhibited by either fluoroquinolone suggesting that hOCT2 was specifically involved in the inhibition. The apparent IC(50) value for the inhibition of [(14)C]creatinine uptake in hOCT2-HEK293 cells was 1.29+/-0.23 microM for DX-619 and 127+/-27 microM for LVFX, indicating DX-619 to be approximately 100-fold more potent than LVFX at inhibiting the transport of [(14)C]creatinine by hOCT2. A Dixon plot revealed that the inhibition by DX-619 of the hOCT2-mediated transport of [(14)C]creatinine was competitive. Fluoroquinolone antibacterials have the ability to inhibit the transport of creatinine by hOCT2, with DX-619 being much more effective than LVFX.

Animals↗

PubMed2005

Regulation of human peptide transporter 1 (PEPT1) in gastric cancer cells by anticancer drugs.

Human peptide transporter 1 (PEPT1) mediates the cellular uptake of di- and tripeptides and peptide-like drugs in the small intestine. In the present study, we examined the regulation of PEPT1 by anticancer drugs in the gastric cancer cell line MKN45. PEPT1 was expressed and functioned in MKN45 cells. The transport activity and mRNA expression of the facilitative glucose transporter 1 (GLUT1) were significantly decreased by 5-fluorouracil treatment, but those of PEPT1 were slightly increased. Cisplatin treatment affected neither PEPT1 nor GLUT1 activity. In conclusion, PEPT1 expressed in MKN45 cells are resistant against the cellular injury induced by 5-fluorouracil and cisplatin.

Antimetabolites, Antineoplastic↗

PubMed2005

Modulation of P-glycoprotein expression in hyperthyroid rat tissues.

P-glycoprotein (Pgp) is expressed in various normal tissues and plays an important role in drug absorption and disposition. In addition, it is supposed that alterations in the expression levels of Pgp are involved in the inter- and intraindividual variability of pharmacokinetics of many drugs. Since pharmacokinetic properties of various drugs are altered in patients with thyroid disorders, we examined the expression of Pgp and mdr1a/1b mRNA in the kidney, liver, jejunum, and ileum from euthyroid and hyperthyroid rats. Western blot analysis revealed that Pgp expression was markedly increased in the kidney and liver of hyperthyroid rats. In contrast, it was slightly increased in the jejunum and ileum. mdr1a/1b mRNA levels were significantly increased in the kidney of hyperthyroid rats. However, they were not increased in the liver as well as in the jejunum and ileum of hyperthyroid rats. Expression levels of bile salt export pump and mdr2 mRNA were also unchanged in hyperthyroid rat liver. Taken together, these findings suggest that thyroid hormone induces Pgp expression in a tissue-selective manner, and that the modulation of mdr1a/1b mRNA expression in the hyperthyroid state varies among tissues.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Restored expression and activity of organic ion transporters rOAT1, rOAT3 and rOCT2 after hyperuricemia in the rat kidney.

We previously reported that in hyperuricemic rats, renal impairment occurred and organic ion transport activity decreased, accompanied with a specific decrease in the expression of rat organic anion transporters, rOAT1 and rOAT3, and organic cation transporter, rOCT2. In the present study, we investigated the reversibility of the organic ion transport activity and expression of organic ion transporters (slc22a) during recovery from hyperuricemia. Hyperuricemia was induced by the administration of a chow containing uric acid and oxonic acid, an inhibitor of uric acid metabolism. Four days after discontinuance of the chow, the plasma uric acid concentration returned to the normal level, and renal functions such as creatinine clearance and BUN levels were restored, although the recovery of tubulointerstitial injury was varied in sites of the kidney. Basolateral uptake of p-aminohippurate (PAH) and tetraethylammonium (TEA), and both protein and mRNA levels of rOAT1, rOAT3 and rOCT2 in the kidney gradually improved during 14 days of recovery from hyperuricemia. Basolateral PAH transport showed a higher correlation with the protein level of rOAT1 (r(2)=0.80) than rOAT3 (r(2)=0.34), whereas basolateral TEA transport showed a strong correlation with rOCT2 protein (r(2)=0.91). The plasma testosterone concentration, which is a dominant factor in the regulation of rOCT2, was gradually restored during the recovery from hyperuricemia, but the correlation between the plasma testosterone level and rOCT2 protein expression in the kidney was not significant. These results suggest that the regulation of organic ion transporters, rOAT1, rOAT3 and rOCT2, by hyperuricemia is reversible, and the organic ion transport activity restores according to the expression levels of these transporters.

Animals↗

PubMed2005

Altered pharmacokinetics of paclitaxel in experimental hepatic or renal failure.

PURPOSE: The aim of this study was to investigate the effect of hepatic or renal insufficiency on the pharmacokinetics of paclitaxel in rats. METHODS: Rats were treated with carbon tetrachloride (CCl4; 0.5 ml/kg) to induce hepatic failure or were subjected to 5/6 nephrectomy (5/6 Nx) to induce renal failure. Paclitaxel (3 mg/kg) was administered intravenously or intraportally. Testosterone 6beta-hydroxylase activity, which is a marker of CYP3A activity, was measured in rat liver microsomes from CCl4-treated or 5/6 Nx rats. RESULTS: After paclitaxel was administered intravenously, total body clearance was significantly reduced by 73% and 34% relative to each control value in CCl4-treated and 5/6 Nx rats, respectively (control, 1.82+/-0.42 vs. CCl4-treated, 0.49+/-0.11; sham, 1.54+/-0.07 vs. 5/6 Nx, 1.01+/-0.12 L h(-1) kg(-1); mean+/-SE, n = 5 to 6). Testosterone 6beta-hydroxylase activity was reduced by 92% and 59% relative to each control value in rat liver microsomes from CCl4-treated and 5/6 Nx rats, respectively. After the intraportal administration of paclitaxel, apparent clearance was reduced by 85% relative to control value in rats with hepatic failure, while that in rats with renal failure was the same as the reduction in systemic clearance. CONCLUSIONS: These results suggested that not only hepatic failure but also renal failure could modify the pharmacokinetics of paclitaxel in vivo.

Animals↗

PubMed2005

Metformin transport by renal basolateral organic cation transporter hOCT2.

PURPOSE: Metformin, an antihyperglycemic agent, is eliminated by tubular secretion in addition to glomerular filtration in the human kidney. This study was performed to characterize metformin transport by human organic cation transporter 2 (hOCT2), the most abundant organic cation transporter in the basolateral membranes of the human kidney. METHODS: Accumulation of [14C]metformin was assessed by the tracer experiments in the human embryonic kidney (HEK293) cells expressing hOCT2. RESULTS: The transport of [14C]metformin was markedly stimulated in hOCT2-expressing cells compared with the vector-transfected cells. The accumulation of [14C]metformin was concentrative and was dependent on the membrane potential, showing consistency with the characteristics of hOCT2. The apparent Km and Vmax values of [14C]metformin transport by hOCT2-expressing HEK293 cells were 1.38+/-0.21 mM and 11.9+/-1.5 nmol mg protein(-1) min(-1), respectively. The order of the potencies of unlabeled biguanides to inhibit [14C]metformin transport by hOCT2 was phenformin > buformin > metformin. Furthermore, [14C]metformin transport was inhibited slightly or moderately by cationic drugs such as procainamide and quinidine at respective therapeutic concentrations. CONCLUSIONS: Metformin is transported by the basolateral organic cation transporter hOCT2 in the human kidney. hOCT2 could play a role in the drug interactions between metformin and some cationic drugs.

Biological Transport↗

PubMed2005

Transcellular transport of creatinine in renal tubular epithelial cell line LLC-PK1.

BACKGROUND/AIM: Creatinine is excreted into urine via tubular secretion in addition to glomerular filtration. In the present study, characteristics of the creatinine transport in renal epithelial cells were investigated. METHODS: The transcellular transport and accumulation of [14C]creatinine and [14C]tetraethylammonium (TEA) were assessed using LLC-PK1 cell monolayers cultured on porous membrane filters. RESULTS: [14C]Creatinine was transported directionally from the basolateral to apical side of LLC-PK1 cell monolayers. Basolateral uptake of [14C]creatinine was dependent on membrane potential, and was saturable with apparent K(m) and V(max) values of 13.2+/-2.8 mM and 13.1+/-3.1 nmol/mg protein/5 min, respectively. Concomitant administration of organic cations (1 mM) such as cimetidine, quinidine and trimethoprim inhibited both the transcellular transport and accumulation of [14C]creatinine. Furthermore, apical excretion of [14C]creatinine was not dependent on acidification of the apical medium. CONCLUSIONS: Creatinine was subjected to directional transport across renal epithelial cells from the basolateral to apical side. The organic cation transporter should be involved in the basolateral uptake of creatinine.

Animals↗

PubMed2005

Metformin is a superior substrate for renal organic cation transporter OCT2 rather than hepatic OCT1.

Although metformin, a cationic agent for type II diabetes, shows its pharmacological effect in the liver, the drug is mainly eliminated into urine. The tissue selectivity based on the function of drug transporters is unclear. In the present study, the transport of metformin was examined using HEK293 cells transiently transfected with five human renal organic ion transporter cDNAs. Human OCT1 and OCT2, but not OAT1, OAT3 or OCT2-A, stimulated the uptake. A kinetic analysis of metformin transport demonstrated that the amount of plasmid cDNA for transfection was also important parameter to the quantitative elucidation of functional characteristics of transporters, and both human and rat OCT2 had about a 10- and 100-fold greater capacity to transport metformin than did OCT1, respectively. In male rats, the mRNA expression level of rOCT2 in the whole kidneys was 8-fold greater than that of rOCT1 in the whole liver. The in vivo distribution of metformin in rats revealed that the expression level of renal OCT2 was a key factor in the control of the concentrative accumulation of metformin in the kidney. These findings suggest that metformin is a superior substrate for renal OCT2 rather than hepatic OCT1, and renal OCT2 plays a dominant role for metformin pharmacokinetics.

Animals↗

PubMed2004

Transient up-regulation of P-glycoprotein reduces tacrolimus absorption after ischemia-reperfusion injury in rat ileum.

Ischemia-reperfusion injury is an unavoidable problem for organ transplantation including small bowel transplantation, and causes a large intra-individual variation of tacrolimus (FK506) pharmacokinetics. Little information is available about the regulation of the intestinal P-glycoprotein expression during tissue regeneration. In the present study, we have examined the molecular and functional variations of ileum P-glycoprotein using rats after ischemia-reperfusion treatment. Morphological study revealed a rapid regeneration of the intestinal wall during 24 h after reperfusion. A reverse transcription-coupled competitive PCR and Western blot analysis revealed that the intestinal expression of P-glycoprotein recovered with time after reperfusion. At 24 h after reperfusion, the ileum P-glycoprotein level was transiently increased to two-fold, and the absorption rate of dihydro-[(3)H]FK506 from in situ ileum loop into portal vein was markedly low in comparison with the control. P-glycoprotein was detected in the crypt area as well as in villous cells at 6 h after reperfusion, and then localized to the apical surface at 24 h consistent with the cell proliferation and differentiation. However, the P-glycoprotein level returned to normal at 48 h. The intra-individual variation in the absorptive rate of tacrolimus was suggested to be regulated by the morphological status of the intestinal epithelium and enterocyte expression level of P-glycoprotein. Therefore, the monitoring of the enterocyte P-glycoprotein level would provide useful information for determining the dosage of tacrolimus immediately after small bowl transplantation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Increased protein level of PEPT1 intestinal H+-peptide cotransporter upregulates absorption of glycylsarcosine and ceftibuten in 5/6 nephrectomized rats.

In chronic renal failure (CRF), dietary protein is one of the factors that deteriorates residual renal functions. Numerous studies have indicated that the products of protein digestion are mainly absorbed as small peptides. However, how small peptides are absorbed in CRF remains poorly understood. H(+)-coupled peptide transporter (PEPT1/SLC15A1) plays an important role in the absorption of small peptides and peptide-like drugs in the small intestine. Because dietary protein intake is one of the risk factors for renal failure, the alteration of intestinal PEPT1 might have implications in the progression of renal disease as well as the pharmacokinetics of peptide-like drugs. In this study, we examined the alteration of intestinal PEPT1 in 5/6 nephrectomized (5/6 NR) rats, extensively used as a model of chronic renal failure. Absorption of [(14)C]glycylsarcosine and ceftibuten was significantly increased in 5/6 NR rats compared with sham-operated rats, without a change in intestinal protease activity. Western blot analysis indicated that the amount of intestinal PEPT1 protein in 5/6 NR rats was increased mainly at the upper region. On the other hand, the amount of intestinal PEPT1 mRNA was not significantly different from that of sham-operated rats. These findings indicate that the increase in absorption of small peptides and peptide-like drugs, caused by the upregulation of intestinal PEPT1 protein, might contribute to the progression of renal failure as well as the alteration of drug pharmacokinetics.

Animals↗

PubMed2004

Different transport properties between famotidine and cimetidine by human renal organic ion transporters (SLC22A).

Histamine H2 receptor antagonist famotidine and cimetidine are commonly used for treatment of gastrointestinal ulcer diseases. Inasmuch as these drugs are mainly secreted by renal tubules, dosages have been adjusted according to renal function. Although many studies have been performed on the molecular mechanisms of renal handling of cimetidine, little is known about that of famotidine. In this study, to examine the recognition and transport of famotidine by human organic anion transporters (OATs; hOAT1, hOAT3) and human organic cation transporter (OCT; hOCT2), the uptake studies using Xenopus laevis oocytes were performed in comparison with cimetidine. The half-maximal inhibitory concentrations of famotidine for [3H]estrone sulfate transport by hOAT3 and [14C]tetraethylammonium transport by hOCT2 (300 microM and 1.8 mM, respectively) were higher than those of cimetidine (53 and 67 microM, respectively). While cimetidine inhibited p-[14C]aminohippurate transport by hOAT1 in a concentration dependent manner, famotidine did not affect it at 5 mM. In addition, hOAT3 mediated famotidine uptake, but hOAT1 and hOCT2 did not show famotidine transport. These results indicate that there are marked differences between famotidine and cimetidine in the recognition and transport by organic ion transporters and that hOAT3 contributes to the renal tubular secretion of famotidine. Present findings should be useful information to understand the renal handling of famotidine and cimetidine.

Animals↗

PubMed2004

Distinct inhibitory effects of tacrolimus and cyclosporin a on calcineurin phosphatase activity.

We have compared the pharmacodynamic properties of calcineurin inhibitors tacrolimus and cyclosporin A in rats to clarify the different therapeutic drug monitoring strategy of both drugs in a clinical situation. In various tissue extracts, the inhibition of calcineurin activity by cyclosporin A was significantly greater than that by tacrolimus at the same drug concentration (1 microM) in the thymus, heart, liver, spleen, kidney, and testis (p < 0.05). The time profiles of blood concentrations and calcineurin activity in whole blood were examined after single or repeated administration of each drug in rats. A substantial time delay in the inhibition was observed following the single administration of tacrolimus or cyclosporin A, resulting in an anticlockwise hysteresis in the relationship between blood concentrations and calcineurin inhibition in whole blood. In contrast, such a hysteresis loop diminished after the repeated administration of each drug, and the recovery rate of calcineurin activity was greater for the inhibition induced by cyclosporin A than by tacrolimus. Furthermore, tacrolimus produced a comparable inhibition of calcineurin activity in whole blood at lower blood concentrations than cyclosporin A. Overall, the effect compartment model well described the time profiles of calcineurin activity in whole blood after the single and repeated administrations of each drug. These findings suggest that the properties of calcineurin inhibition differ between tacrolimus and cyclosporin A. Distinct pharmacodynamics may partly contribute to the therapeutic drug monitoring strategy in transplant patients receiving calcineurin inhibitors.

Animals↗

PubMed2004

Efflux properties of basolateral peptide transporter in human intestinal cell line Caco-2.

Small peptides and some pharmacologically active compounds are absorbed from the small intestine by the apical H(+)-coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter. Here we investigated the efflux properties of the basolateral peptide transporter in Caco-2 cells using two strategies, efflux measurements and a kinetic analysis of transepithelial transport of glycylsarcosine (Gly-Sar). [(14)C]Gly-Sar efflux through the basolateral membrane was not affected significantly by the external pH. Both approaches revealed that the basolateral peptide transporter was saturable in the efflux direction, and that the affinity was lower than that in the influx direction. For two peptide-like drugs, there was no difference in substrate recognition by the basolateral peptide transporter between the two sides of the membrane. Using the kinetic parameters of PEPT1 and the basolateral peptide transporter, a computational model of Gly-Sar transport in Caco-2 cells was constructed. The simulation fitted the experimental data well. Our findings suggested that substrate affinity of the basolateral peptide transporter is apparently asymmetric, but pH-dependence and substrate specificity are symmetric for the two directions of transport. The behaviour of Gly-Sar in Caco-2 cells could be predicted by a mathematical model describing the peptide transporters.

Anti-Bacterial Agents↗

PubMed2004

Genetic variant Arg57His in human H+/peptide cotransporter 2 causes a complete loss of transport function.

We evaluated the functional consequences of genetic variations in human H(+)/peptide cotransporter 2 (hPEPT2, SLC15A2) resulting in the amino acid changes Arg57His (R57H) and Pro409Ser (P409S). The transport activity of variant R57H was completely abolished, whereas that of variant P409S was comparable with that of wild-type hPEPT2 at pH 5.0-8.0. R57H variant protein was detected in the crude membranes of transiently expressed HEK293 cells by Western blot analysis. The expression of the R57H variant at the plasma membrane was confirmed by indirect immunofluorescence in Xenopus oocytes, suggesting that the loss of transport function of hPEPT2 R57H was not due to a change in membrane protein expression. This is the first demonstration of a functional impairment of the SLC15A family induced by a single nucleotide polymorphism.

Amino Acid Substitution↗

PubMed2004

Lansoprazole-tacrolimus interaction in Japanese transplant recipient with CYP2C19 polymorphism.

OBJECTIVE: To report a patient with a high tacrolimus blood concentration after lansoprazole administration and assess the potential interaction between tacrolimus and lansoprazole. CASE SUMMARY: A 34-year-old Japanese man underwent a living-donor kidney transplantation having received tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression. Lansoprazole was administered from postoperative day 4 as prophylaxis of ulcers. The trough concentration of tacrolimus increased markedly after the introduction of lansoprazole, while results of liver function tests were within normal limits. Lansoprazole was stopped on day 15 and was replaced with famotidine on day 17. The trough concentration of tacrolimus returned to the therapeutic range after administration of lansoprazole ceased. Genetic analysis revealed a heterozygous mutation at exon 5 of the CYP2C19 gene (CYP2C19*1/*2) in this patient. DISCUSSION: Lansoprazole is metabolized by 2 enzymes, CYP2C19 and CYP3A4. Since tacrolimus is also metabolized by CYP3A4, the blood concentration of tacrolimus in this patient who had a CYP2C19 gene mutation may have been elevated by decreased hepatic elimination of lansoprazole. An objective causality assessment revealed that this interaction was probable. CONCLUSIONS: Trough concentrations of tacrolimus should be monitored closely for optimizing the dosage regimen in patients receiving concomitant lansoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2004

A non-immunological phospholipid-dependent coagulation inhibitor associated with IgGlambda-type multiple myeloma.

We investigated the rare case of a patient with IgGlambda multiple myeloma for whom both prothrombin time and APTT were significantly prolonged. The IgG inhibited coagulation reactions upstream from prothrombin when coagulation was initiated by mRVVT, but not by FXa, as indicated by a chromogenic substrate for FXa. The mPT and the mAPTT showed inhibition of FXa generation in both the intrinsic and extrinsic pathways. The IgG inhibited both protein C (indicated by APTT) and FX (indicated by RVV) but not amidolysis for either activated protein C or FXa. The addition of excess phospholipid significantly shortened the prolonged RVVT of the patient. It inhibited the coagulation reactions of normal plasma and was dependent on decreasing the PS concentration in the APTT reagent. It was suggested that the IgG showed lupus anticoagulant (LA)-like activity that inhibited phospholipid-dependent coagulation reactions in the intrinsic, extrinsic, and common pathways. However, the IgG did not bind cardiolipin-beta2GPI complex, beta2GPI, or prothrombin in ELISA assays. The IgG did not bind to either PS or phospholipid complexes in the presence or absence of prothrombin, FX, or FXa. Interestingly, the IgG lost its LA like-activity when it was degraded to F(ab')2 and Fc fragments by pepsin. We suspected that the IgG might inhibit the interaction between coagulation factors and acid phospholipid non-immunologically and that this process requires an intact IgG conformation, although the reaction mode is still not clear.

Blood Coagulation↗