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Biomedical subjects

K Hosoya

Publications and source records attributed to K Hosoya.

At least 19 recordsLinked to original sources
PubMed2006

Plasma leptin concentration in dogs: effects of body condition score, age, gender and breeds.

Leptin is a cytokine produced by adipocytes, and plays a key role in the regulation of energy balance. In the present study, we measured plasma leptin concentrations of 166 normal and obese dogs visiting veterinary practices, and clarified the influence of age, gender and breed on plasma leptin levels in dogs. Leptin levels were higher in the dogs with higher body condition scores. There was no noticeable influence of age, gender and breed, but those in optimal puppies and obese Miniature Dachshund tended to be lower than those in corresponding groups. We conclude that plasma leptin is a reliable marker of adiposity in dogs regardless of age, gender and breed variations, and thereby useful as a blood biochemistry test for health examinations and treatment of obesity.

Aging↗
PubMed2003

Powdery mildew (Sphaerotheca fuliginea) resistance in melon is selectable at the haploid level.

The major cause of powdery mildew in melons (Cucumis melo L.) is the fungus Sphaerotheca fuliginea. There are several cultivar- and season-specific races of this fungus. In order to control powdery mildew, it is important to introduce resistance to fungal infection into new cultivars during melon breeding. Haploid breeding is a powerful tool for the production of pure lines. In this study, it was investigated whether powdery mildew resistance could be manifested at the haploid level from two disease-resistant melon lines, PMR 45 and WMR 29. the effects of various races of S. fuliginea on diploid and haploid plants of PMR 45 and WMR 29 and of a disease-susceptible line, Fuyu 3 were measured. The responses of haploid and diploid plants to powdery mildew were identical. In addition, haploids that were generated from hybrids between Fuyu 3 and disease-resistant lines were examined. Seven out of 13 haploids from a Fuyu 3xPMR 45 cross and 10 out of 12 haploids from a Fuyu 3xWMR 29 cross were classified as resistant plants because they showed the same responses as their disease-resistant diploid parents to the various fungal races. These results indicate that resistance in PMR 45 and WMR 29 is selectable at the haploid level. All of the plant responses were observed by microscopy. A possible mechanism for generating powdery mildew resistance in two different melon lines is discussed.

Cucumis melo↗
PubMed2001

Preparation strategy for uniformly sized, polymer-based HPLC packing materials having practically acceptable column efficiency. 1. Copolymerization technique.

Uniformly sized, polymer-based packing materials affording excellent chromatographic performance in semimicro HPLC were prepared from mixtures of alkyl methacrylate and glycerol dimethacrylate by a copolymerization technique using a multistep swelling and polymerization method. A column efficiency of up to 13000 plates was obtained for a 2-mm-i.d. x 150-mm column with beads prepared from a 1:1 (v:v) mixture of an alkyl methacrylate and glycerol dimethacrylate. The packing materials exhibit a considerable improvement in column efficiency, as demonstrated on the separation of polyaromatic hydrocarbons (PAHs) having a relatively long retention time, which thus far has been a serious problem of polymer-based packing materials. Detailed studies suggested a rather limited optimum pore size, and its distribution was depicted to get the excellent column efficiency on polymer-based packing materials.

Journal Article↗
PubMed2001

Selectivity of stationary phases in reversed-phase liquid chromatography based on the dispersion interactions.

Selectivity of 15 stationary phases was examined, either commercially available or synthesized in-house. The highest selectivity factors were observed for solute molecules having different polarizability on the 3-(pentabromobenzyloxy)propyl phase (PBB), followed by the 2-(1-pyrenyl)ethyl phase (PYE). Selectivity of fluoroalkane 4,4-di(trifluoromethyl)-5,5,6,6,7,7,7-heptafluoroheptyl (F13C9) phase is lowest among all phases for all compounds except for fluorinated ones. Aliphatic octyl (C8) and octadecyl (C18) phases demonstrated considerable selectivity, especially for alkyl compounds. While PBB showed much greater preference for compounds with high polarizability containing heavy atoms than C18 phase, F13C9 phase showed the exactly opposite tendency. These three stationary phases can offer widely different selectivity that can be utilized when one stationary phase fails to provide separation for certain mixtures. The retention and selectivity of solutes in reversed-phase liquid chromatography is related to the mobile phase and the stationary phase effects. The mobile phase effect, related to the hydrophobic cavity formation around non-polar solutes, is assumed to have a dominant effect on retention upon aliphatic stationary phases such as C8, C18. In a common mobile phase significant stationary phase effect can be attributed to dispersion interaction. Highly dispersive stationary phases such as PBB and PYE retain solutes to a significant extent by (attractive) dispersion interaction with the stationary phase ligands, especially for highly dispersive solutes containing aromatic functionality and/or heavy atoms. The contribution of dispersion interaction is shown to be much less on C18 or C8 phases and was even disadvantageous on F13C9 phase. Structural properties of stationary phases are analyzed and confirmed by means of quantitative structure-chromatographic retention (QSRR) study.

Chromatography, Liquid↗
PubMed2001

Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat.

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 approximately 9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33 degrees C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39 degrees C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O -methyl- D -glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 +/- 0.51 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus).

We have isolated a cDNA for Cm-HMGR, encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in melon (Cucumis melo L. reticulatus; Genbank Accession No. AB021862). Cm-HMGR encodes a polypeptide of 588 amino acids that contains two transmembrane domains and a catalytic domain. Database searches revealed that Cm-HMGR shows homology to HMG1 (63.7%) and HMG2 (70.3%) of tomato, to HMG1 (77.2%) and HMG2 (69.4%) of Arabidopsis thaliana, and to HMGR of tobacco (72.6%). Functional expression in a HMG-CoA reductase-deficient mutant yeast showed that Cm-HMGR products mediate the synthesis of mevalonate. Northern analysis revealed that the level of Cm-HMGR mRNA in the fruit increased after pollination and markedly decreased at the end of fruit enlargement. During ripening, Cm-HMGR mRNA levels increased markedly in the fruit. In parallel with mRNA expression, Cm-HMGR activity increased after pollination, whereas no Cm-HMGR activity was detectable during fruit ripening. Our results suggest that Cm-HMGR is important during early post-pollination development of the fruit in melon.

Amino Acid Sequence↗
PubMed2001

Chromosomal sublocalization and microsatellite DNA polymorphism of human adrenomedullin gene.

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues such as the heart, kidney and vascular cells. We have cloned and sequenced genomic DNA encoding the human AM gene. In this study, we determined that the AM gene was located in the short arm of chromosome 11 (p15.1-3). The 3'-end of the gene is flanked by a microsatellite marker of cytosine adenine (CA) repeats. Moreover, we analyzed this DNA variation in the AM gene in the general Japanese population. Genomic DNA was obtained from the peripheral leukocytes of healthy normotensive subjects, 327 men and 149 women, aged 51 +/- 8 years (mean +/- SD). The genomic DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined via polyacrylamide gel electrophoresis (PAGE). Plasma AM concentration was measured by RIA and compared with respect to the number of CA repeats adjacent to the AM gene. In Japanese, four types of alleles with different CA-repeat numbers; 11, 13, 14 and 19, appear to exist. The frequencies of these alleles were as follows: 11 repeats, 28.8%; 13 repeats, 33.1%; 14 repeats, 35.0% and 19 repeats, 3.1%. This DNA variation does not seem to affect the transcription of the AM gene, because plasma concentrations of AM were not significantly different between the genotypes.

Adrenomedullin↗
PubMed2001

Characterization of the amino acid transport of new immortalized choroid plexus epithelial cell lines: a novel in vitro system for investigating transport functions at the blood-cerebrospinal fluid barrier.

PURPOSE: To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). METHODS: Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. RESULTS: Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. CONCLUSIONS: Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

Amino Acids↗
PubMed2001

MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina.

PURPOSE: The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB. METHODS: TR-iBRB2 cells were cultured at 33 degrees C, and L-lactic acid uptake was monitored by measuring [14C]L-lactic acid at 37 degrees C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively. RESULTS: The [14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [14C]L-lactic acid uptake at pH 6.0 was both temperature- and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxyphenylhydrazone (protonophore), alpha-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H+-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H+ gradient. [14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na+/H+ exchanger I mRNA was expressed in TR-iBRB2 cells. CONCLUSION: L-Lactic acid transport at the iBRB is an H-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.

Animals↗
PubMed2001

Microsatellite DNA polymorphism of human adrenomedullin gene in normotensive subjects and patients with essential hypertension.

Adrenomedullin (AM) is a hypotensive peptide widely produced in the cardiovascular organs and tissues. We have cloned and sequenced the genomic DNA encoding the human AM gene and have determined that the gene is located in the short arm of chromosome 11. The 3'-end of the gene is flanked by the microsatellite marker of cytosine adenine (CA) repeats. In this study, we investigated the association between DNA variations in AM gene and the predisposition to hypertension. Genomic DNA was obtained from 272 healthy normotensive subjects (NT) age 57+/-5 years and 266 patients with essential hypertension (EH) age 53+/-11 years. The DNA was subject to PCR using a fluorescence-labeled primer, and the number of CA repeats were determined by poly-acrylamide gel electrophoresis. The averaged blood pressure was 117+/-13/73+/-9 mm Hg in NT and 170+/-23/104+/-12 mm Hg in EH. In Japanese, there existed 4 types of alleles with different CA-repeat numbers: 11, 13, 14, and 19. The frequencies of these alleles were significantly different between NT and EH (chi(2)=9.43, P=0.024). Namely, 13.5% of EH carried the 19-repeat allele, whereas the frequency was 6.2% in NT (chi(2)=7.62, P=0.007). In NT, plasma AM concentrations were not significantly different between the genotypes. In conclusion, microsatellite DNA polymorphism of AM gene may be associated with the genetic predisposition to EH, although the gene expression is not likely to be affected by the genotypes.

Adrenomedullin↗
PubMed2001

Conditionally immortalized cell lines as a new in vitro model for the study of barrier functions.

Conditionally immortalized brain and retinal capillary endothelial and choroid plexus epithelial cell lines were established from a transgenic rat (Tg rat) and mouse (Tg mouse) harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen. These cell lines exhibit temperature-sensitive cell growth due to the expression of ts SV 40 large T-antigen. Mouse brain (TM-BBB) and rat brain (TR-BBB) and rat retinal (TR-iBRB) capillary endothelial cell lines appear to have a spindle-fiber shaped morphology and exhibit the typical endothelial markers, such as von Willebrand factor and acetylated low-density lipoprotein uptake. These cell lines express in vivo influx and efflux transporters, such as P-glycoprotein (P-gp) and GLUT1, which is capable of 3-O-methyl-D-glucose transport. TM-BBB cells are able to undergo efflux transport of cyclosporin A, which is a substrate for P-gp transport activity. They may also express oatp2 and exhibit dehydroepiandrosterone sulfate and digoxin uptake activity. TR-BBB cells express the mRNA of multidrug resistance associated protein 1 (MRP1) and a large neutral amino acid transporter, which consists of LAT1 and 4F2hc. TR-iBRB cells exhibit pH-dependent L-lactic acid transport activity and express the mRNA of monocarboxylate transporter (MCT) 1 and 2. The choroid plexus epithelial cell line (TR-CSFB) has polygonal cell morphology, expresses the typical choroid plexus epithelial cell marker, transthyretin, and has Na+, K+-ATPase located on the apical side. TR-CSFB cells also exhibit amino acid transport activity which has been observed in vivo. These barrier cell lines established from the Tg rat and Tg mouse have in vivo transport functions and are good in vitro models for drug transport to the brain and retina and as a screen for drugs which might be capable of delivery to the brain and retina.

Animals↗
PubMed2000

Brain-to-blood efflux transport of estrone-3-sulfate at the blood-brain barrier in rats.

Efflux transport of estrogens such as estrone-3-sulfate (E1S), and estrone (E1) across the blood-brain barrier (BBB) was evaluated using the Brain Efflux Index (BEI) method. The apparent BBB efflux rate constant (Keff) of [3H]E1S, and [3H]E1 was 6.63 x 10(-2) +/- 0.77 x 10(-2) min(-1), and 6.91 x 10(-2) +/- 1.23 x 10(-2) min(-1), respectively. The efflux transport of [3H]E1S from brain across the BBB was a saturable process with Michaelis constant (Km) of 96.0 +/- 34.4 microM and 93.4 +/- 22.0 microM estimated by two different methods. By determining [3H]E1S metabolites using high performance liquid chromatography (HPLC) after intracerebral injection, significant amounts of [3H]E1S were found in the jugular venous plasma, providing direct evidence that most of [3H]E1S is transported from brain across the BBB in intact form. To compare the apparent efflux clearance across the BBB of E1S with that of E1, the brain distribution volume of E1S and E1 was estimated using the brain slice uptake method. The apparent efflux clearance of [3H]E1S was determined to be 74.9 +/- 3.8 microl/(min x g brain) due to the distribution volume of 1.13 +/- 0.06 ml/g brain. By contrast, the apparent efflux clearance of E1 was more than 227 +/- 3 microl/(min x g brain), since the distribution volume of [3H]E1 at 60 min was 3.28 +/- 0.13 ml/g. The E1S efflux transport process was inhibited by more than 40% by coadministration of bile acids (taurocholate, and cholate), and organic anions (sulfobromophthalein, and probenecid), whereas other organic anions did not affect the E1S efflux transport. The [3H]E1S efflux was significantly reduced by 48.6% after preadministration of 5 mM dehydroepiandrosterone sulfate. These results suggest that E1S is transported from brain to the circulating blood across the BBB via a carrier-mediated efflux transport system.

Animals↗
PubMed2000

Stationary phase structures enhancing electroosmotic flow in capillary electrochromatography.

Several chemically bonded silicas with C18 groups were examined with respect to electroosmotic flow (EOF) velocities under CEC conditions. Stationary phases with low hydrophobic selectivity generally provided high EOFs. The stationary phases prepared by using octadecyltrichlorosilane showed greater EOF than those from octadecyldimethylchlorosilane. Restricted-access reversed-phase (RARP) packing materials having C18 groups inside the pores and silanols on the external surfaces showed higher EOF than monomeric C18 phases with similarly high hydrophobic selectivity. The RARP-type structure having silanols at the external surface seems to be effective for increasing EOF while maintaining the hydrophobic character of the solute binding sites.

Electrophoresis, Capillary↗
PubMed2000

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.

Journal Article↗
PubMed2000

Force-length relationship in dogs as a measure of protective effect of imidapril on regional myocardial ischemia and reperfusion injury.

Our laboratory previously reported that the end-systolic force-length relationship of the left ventricle provides a better method of evaluating myocardial contractile properties than the left ventricular end-systolic pressure-volume relationship, because it avoids deficiencies of the latter parameter such as dependence of its slope (E(max)) on the volume intercept (V(0)). The slope (E(c)) of the left ventricular end-systolic force-length relationship represents the contractility of functioning myocardium, while its length intercept (L(0)) reflects the length of non-functioning myocardium. However, the effect of regional myocardial ischemia on these parameters, as evaluated by the force-length relationship, remains unknown. To clarify the effects of regional ischemia and angiotensin-converting enzyme inhibition on the myocardium during ischemia-reperfusion, the changes in E(c) and L(0) were determined in anesthetized open-chest dogs. (1) Control group (n=26): Before and after 15 min of complete coronary artery occlusion, as well as after 15 min of reperfusion, left ventricular pressure and volume were simultaneously recorded during inferior vena cava occlusion. The left ventricular force-length relationship was obtained from the pressure and volume of three cylindrical segments of the ventricle, and E(c) and L(0) were calculated. (2) Imidapril group (n=14): Imidaprilat (1 microg/kg/min) was continuously infused from 30 min before ischemia to the end of the experiment, and the same procedures were followed as in the control group. Fourteen out of the 26 dogs (54%) in the control group died of reperfusion-induced ventricular arrhythmias, while only two of the 14 dogs (14%) in the imidapril group did so (P<0.05). In the control group, E(c) was increased during ischemia and remained at the same level after reperfusion. However, E(c) was not altered in the imidapril group. Although L(0) was increased during ischemia and decreased after reperfusion in both groups, the percent increase of L(0) in the imidapril group was significantly smaller than in the control group (8% vs. 32%, P<0.05). With the improvement of these indices, the bradykinin concentration of coronary venous blood increased in the imidapril group (P<0.01). These findings suggest that regional myocardial ischemia increased the average contractility of overall functioning myocardium despite the increased non-functioning myocardium. Moreover, imidapril has a cardioprotective effect against ischemia-reperfusion injury by decreasing infarct size, and through the antiarrhythmic effect and the reversal of increased overall contractility.

Adrenergic beta-Antagonists↗
PubMed2000

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2.

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.

Animals↗