The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Tetsuya Terasaki - One of the best experts on this subject based on the ideXlab platform.
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functional expression of a proton coupled organic cation h oc antiporter in human Brain Capillary endothelial cell line hcmec d3 a human blood Brain barrier model
Fluids and Barriers of the CNS, 2013Co-Authors: Keita Shimomura, Tetsuya Terasaki, Pierre-olivier Couraud, Takashi Okura, Sayaka Kato, Jeanmichel Schermann, Yoshiharu DeguchiAbstract:Background Knowledge of the molecular basis and transport function of the human blood–Brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human Brain Capillary endothelial cells, because human Brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human Brain Capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.
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quantitative proteomics of transporter expression in Brain Capillary endothelial cells isolated from p glycoprotein p gp breast cancer resistance protein bcrp and p gp bcrp knockout mice
Drug Metabolism and Disposition, 2012Co-Authors: Sagar Agarwal, Tetsuya Terasaki, Yasuo Uchida, Rajendar K Mittapalli, Ramola Sane, William F ElmquistAbstract:The objective of this study was to quantitatively examine the protein expression of relevant transporters and other proteins in the Brain Capillary endothelial cells isolated from wild-type mice and P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. After the isolation of Brain Capillary endothelial cells, a highly sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was used to determine the quantitative expression of membrane transporters at the blood-Brain barrier (BBB) of the various mouse genotypes. Quantitative expression of 29 protein molecules, including 12 ATP-binding cassette transporters, 10 solute carrier transporters, five receptors, and two housekeeping proteins, was examined by quantitative proteomics in the four mouse genotypes. There was no significant difference in the expression of P-gp between the wild-type and Bcrp1(−/−) mice. Likewise, Bcrp expression was not significantly different between the wild-type and Mdr1a/b(−/−) mice. There was no significant difference in the expression of any of the measured proteins in the Brain Capillary endothelial cells across the genotypes, except for the lack of expression of the corresponding protein in the mice that had a genetic deletion of P-gp or Bcrp. In conclusion, using a quantitative proteomic approach, we have shown that there are no changes in the expression of several relevant transporters in Brain Capillary endothelial cells isolated from single and combination knockout mice. These data suggest that the mechanism behind the functional compensation between P-gp and Bcrp at the BBB is not related to compensatory changes in transporter expression.
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mRNA expression levels of tight junction protein genes in mouse Brain Capillary endothelial cells highly purified by magnetic cell sorting
Journal of neurochemistry, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Yuki Katsukura, Tomoko Asashima, Tetsuya TerasakiAbstract:Tight junctions (TJs) are an important component of the blood-Brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of Brain Capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 106 cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the Brain Capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-Brain barrier in cooperation with claudin-5.
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establishing a method to isolate rat Brain Capillary endothelial cells by magnetic cell sorting and dominant mrna expression of multidrug resistance associated protein 1 and 4 in highly purified rat Brain Capillary endothelial cells
Pharmaceutical Research, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Tetsuya TerasakiAbstract:To establish a method for isolating highly purified Brain Capillary endothelial cells (BCECs) from rat Brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs. The cells were prepared from the Capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis. From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat Brain. The contamination by neurons and astrocytes was markedly less than in the Brain Capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively. This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-Brain barrier.
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exogenous expression of claudin 5 induces barrier properties in cultured rat Brain Capillary endothelial cells
Journal of Cellular Physiology, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Saori Sato, Mayu Kamoi, Tetsuya TerasakiAbstract:Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-Brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat Brain Capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat Brain Capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat Brain Capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat Brain Capillary fraction (per total mRNA) was 35.6-fold greater than that in whole Brain, while the level of claudin-12 mRNA was only 13.9% of that in whole Brain, suggesting that expression of claudin-12 mRNA is not restricted to Brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell–cell boundaries. The permeability surface product of [14C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 × 10−3 and 11.6 × 10−3 cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in Brain capillaries, and plays a key role in the appearance of barrier properties of Brain Capillary endothelial cells. J. Cell. Physiol. 210: 81–86, 2007. © 2006 Wiley-Liss, Inc.
Christian Frelin - One of the best experts on this subject based on the ideXlab platform.
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characterization and channel coupling of the p2y12nucleotide receptor of Brain Capillary endothelial cells
Journal of Biological Chemistry, 2002Co-Authors: Joseph Simon, Christian Frelin, Alexander K Filippov, Sara A Goransson, Yung Hou Wong, A D Michel, David Brown, Eric A BarnardAbstract:Abstract Rat Brain Capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y12 ADP receptor (“P2Y T”) of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC50, 2 nm), ADP, and adenosine 5′-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-d-β,γ-difluoromethylene-ATP was a competitive antagonist (K B, 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC50, 0.4 nm), ATP (1.9 μm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC50, 0.1 nm) GIRK1/GIRK2 inward rectifier K+ channels when co-expressed with P2Y12 receptors in sympathetic neurons. Surprisingly, P2Y1 receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y12 receptors. A new P2Y12-mediated transduction was found, the closing of native N-type Ca2+channels; again both 2-MeSATP and 2-MeSADP are agonists (EC50, 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca2+ channel inhibition and K+ channel activation are mediated by βγ subunit release from a heterotrimeric G-protein. Gα subunit types in B10 cells were also identified. The presence in the Brain Capillary endothelial cell of the P2Y12 receptor is a significant extension of its functional range.
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PPADS Inhibits P2Y1Purinoceptors in Rat Brain Capillary Endothelial Cells and in Rat Ileal Myocytes by an Indirect Mechanism
Biochemical and biophysical research communications, 1998Co-Authors: Paul Vigne, Jean Philippe Breittmayer, P. Pacaud, Gervaise Loirand, Christian FrelinAbstract:Abstract P2Y 1 receptor-like responses were analyzed in rat ileal myocytes and in rat Brain Capillary endothelial cells. In endothelial cells, pyridoxal phosphate-6-azophenyl-2′,4′disulfonic acid (PPADS) inhibits ADP induced intracellular Ca 2+ transients with a half maximum effect at 3 μM. PPADS shifts ADP dose response curves to larger concentrations. Yet PPADS is inactive when added at the same time as ADP. A preequilibration of the cells with PPADS is necessary to observe its inhibitory action. Similarly in ileal myocytes, PPADS has no action on ADP responses when it is applied at the same time as ADP. Actions of PPADS require a preequilibration with the cells and are fully reversible. These results suggest that PPADS is not a competitive antagonist of P2Y 1 receptors and caution about its usefulness to distinguish subtypes of P2Y 1 receptors.
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Sensitization by calyculin A of Brain Capillary endothelial cells to endothelin‐1
British Journal of Pharmacology, 1995Co-Authors: Paul Vigne, Jean Philippe Breittmayer, Christian FrelinAbstract:1 Cultured Brain Capillary endothelial cells of the rat respond to endothelin-1 (ET-1) by an increased activity of the Na+,K+,2Cl−, cotransporter and a mobilization of intracellular Ca2+ stores. 2 Calyculin A (1–30 nm), but not okadaic acid, sensitizes up to 100 fold the Na+,K+,2Cl− cotransporter to the action of ET-1. 3 Calyculin A (30 nm) does not modify the binding properties of ET-1 to ETA receptors. 4 Calyculin A (30 nm) inhibits ET-1 induced intracellular Ca2+ mobilization. 5 It is concluded that inhibition of protein phosphatase 1 selectively modifies the repertoire of intracellular actions of ET-1 and favours actions that are unrelated to the phospholipase C signalling cascade.
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na k cl cotransporter of Brain Capillary endothelial cells properties and regulation by endothelins hyperosmolar solutions calyculin a and interleukin 1
Journal of Biological Chemistry, 1994Co-Authors: Paul Vigne, Lopez A Farre, Christian FrelinAbstract:Abstract Cultured rat Brain Capillary endothelial cells expressed a large 86Rb+ uptake component that was dependent on external Na+ and Cl- and that was inhibited by loop diuretics with unusual pharmacological properties: benzmetanide (IC50 = 1-5 microM) = bumetanide (IC50 = 1-5 microM) > piretanide (IC50 = 3-16 microM) = furosemide (IC50 = 7-11 microM). It was activated 2-fold by endothelin-1 (EC50 = 1 nM) and endothelin-3 (EC50 = 9 nM). The actions of endothelins were prevented by BQ-123 (cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu)) in a competitive manner and with a high affinity, thus indicating the involvement of an atypical BQ-123-sensitive, ETA-like receptor that had a high affinity for endothelin-3. Neither protein kinase C nor Ca(2+)-dependent protein kinases mediated the actions of endothelins. Cotransport activity was increased 4-fold by hyperosmotic cell shrinkage. Basal Na(+)-K(+)-Cl- cotransport activity was partially inhibited by isoproterenol and was unaffected by agents that promoted cGMP formation. Calyculin A, an inhibitor of protein phosphatases, stimulated cotransport activity and potentiated the action of endothelin-1, but not that of cell shrinkage. Basal and stimulated cotransport activities were inhibited by genistein, a protein kinase inhibitor with similar potencies, and by staurosporine, which has different potencies. Finally, endothelin-1-stimulated activity was partially and specifically inhibited by interleukin-1. It is concluded that rat Brain Capillary endothelial cells express a Na(+)-K(+)-Cl- cotransporter that has unique properties and that is regulated by multiple protein kinase/phosphatase systems. It is a target for low concentrations of endothelins and may play a role in Brain-to-blood movements of K+.
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Endothelins inhibit adenylate cyclase in Brain Capillary endothelial cells.
Biochemical and biophysical research communications, 1991Co-Authors: Annie Ladoux, Christian FrelinAbstract:The action of endothelins (Et) on cAMP formation was studied in endothelial cells from rat Brain microvessels. Et-1 and Et-3 had no action by themselves. They both inhibited cholera toxin stimulated adenylate cyclase by about 50%. K0.5 values were observed at 2 nM and 40 nM for Et-1 and Et-3 respectively, indicating an involvement of a low affinity Et-3 receptor. Coupling to adenylate cyclase was achieved by a pertussis toxin sensitive mechanism. Another action of endothelins in Brain Capillary endothelial cells was to stimulate phospholipase C. This action involved a low affinity Et-3 receptor and a pertussis toxin insensitive mechanism. It is concluded that in Brain Capillary endothelial cells, ETA like receptors are coupled to phospholipase C and to adenylate cyclase via two different mechanisms.
Sumio Ohtsuki - One of the best experts on this subject based on the ideXlab platform.
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Efficient isolation of Brain Capillary from a single frozen mouse Brain for protein expression analysis.
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2020Co-Authors: Seiryo Ogata, Shingo Ito, Takeshi Masuda, Sumio OhtsukiAbstract:Isolated Brain capillaries are essential for analyzing the changes of protein expressions at the blood–Brain barrier (BBB) under pathological conditions. The standard Brain Capillary isolation meth...
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mRNA expression levels of tight junction protein genes in mouse Brain Capillary endothelial cells highly purified by magnetic cell sorting
Journal of neurochemistry, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Yuki Katsukura, Tomoko Asashima, Tetsuya TerasakiAbstract:Tight junctions (TJs) are an important component of the blood-Brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of Brain Capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 106 cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the Brain Capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-Brain barrier in cooperation with claudin-5.
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establishing a method to isolate rat Brain Capillary endothelial cells by magnetic cell sorting and dominant mrna expression of multidrug resistance associated protein 1 and 4 in highly purified rat Brain Capillary endothelial cells
Pharmaceutical Research, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Tetsuya TerasakiAbstract:To establish a method for isolating highly purified Brain Capillary endothelial cells (BCECs) from rat Brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs. The cells were prepared from the Capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis. From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat Brain. The contamination by neurons and astrocytes was markedly less than in the Brain Capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively. This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-Brain barrier.
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exogenous expression of claudin 5 induces barrier properties in cultured rat Brain Capillary endothelial cells
Journal of Cellular Physiology, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Saori Sato, Mayu Kamoi, Tetsuya TerasakiAbstract:Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-Brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat Brain Capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat Brain Capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat Brain Capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat Brain Capillary fraction (per total mRNA) was 35.6-fold greater than that in whole Brain, while the level of claudin-12 mRNA was only 13.9% of that in whole Brain, suggesting that expression of claudin-12 mRNA is not restricted to Brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell–cell boundaries. The permeability surface product of [14C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 × 10−3 and 11.6 × 10−3 cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in Brain capillaries, and plays a key role in the appearance of barrier properties of Brain Capillary endothelial cells. J. Cell. Physiol. 210: 81–86, 2007. © 2006 Wiley-Liss, Inc.
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Localization of norepinephrine and serotonin transporter in mouse Brain Capillary endothelial cells.
Neuroscience research, 2002Co-Authors: Kentaro Wakayama, Hitomi Takanaga, Sumio Ohtsuki, Ken-ichi Hosoya, Tetsuya TerasakiAbstract:Monoamines function as a vasoactive modulator in the central nervous system (CNS) and are believed to regulate blood-Brain barrier (BBB) function. Although monoamine transport is an essential process for regulating the extracellular monoamine concentration, the transport systems for monoamines at the BBB are poorly understood. mRNA expression of norepinephrine transporter (NET) and serotonin transporter (SERT) has been detected in a conditionally immortalized mouse Brain Capillary endothelial cell line (TM-BBB4) used as an in vitro model of the BBB, whereas no dopamine transporter (DAT) was detected. Western blot analysis showed the expression of NET and SERT protein in the membrane fraction of mouse Brain capillaries and TM-BBB4 cells. Immunohistochemical analysis revealed that NET and SERT are localized at the Brain capillaries in the mouse cerebral cortex, and suggests that NET is localized at the abluminal side of Brain Capillary endothelial cells, and SERT is localized at the luminal and abluminal sides. NET and SERT expressed at the BBB may be involved in the inactivation of monoamines released from neurons around the BBB.
Hirofumi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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mRNA expression levels of tight junction protein genes in mouse Brain Capillary endothelial cells highly purified by magnetic cell sorting
Journal of neurochemistry, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Yuki Katsukura, Tomoko Asashima, Tetsuya TerasakiAbstract:Tight junctions (TJs) are an important component of the blood-Brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of Brain Capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 106 cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the Brain Capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-Brain barrier in cooperation with claudin-5.
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establishing a method to isolate rat Brain Capillary endothelial cells by magnetic cell sorting and dominant mrna expression of multidrug resistance associated protein 1 and 4 in highly purified rat Brain Capillary endothelial cells
Pharmaceutical Research, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Tetsuya TerasakiAbstract:To establish a method for isolating highly purified Brain Capillary endothelial cells (BCECs) from rat Brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs. The cells were prepared from the Capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis. From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat Brain. The contamination by neurons and astrocytes was markedly less than in the Brain Capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively. This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-Brain barrier.
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exogenous expression of claudin 5 induces barrier properties in cultured rat Brain Capillary endothelial cells
Journal of Cellular Physiology, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Saori Sato, Mayu Kamoi, Tetsuya TerasakiAbstract:Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-Brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat Brain Capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat Brain Capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat Brain Capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat Brain Capillary fraction (per total mRNA) was 35.6-fold greater than that in whole Brain, while the level of claudin-12 mRNA was only 13.9% of that in whole Brain, suggesting that expression of claudin-12 mRNA is not restricted to Brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell–cell boundaries. The permeability surface product of [14C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 × 10−3 and 11.6 × 10−3 cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in Brain capillaries, and plays a key role in the appearance of barrier properties of Brain Capillary endothelial cells. J. Cell. Physiol. 210: 81–86, 2007. © 2006 Wiley-Liss, Inc.
Tomoko Asashima - One of the best experts on this subject based on the ideXlab platform.
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mRNA expression levels of tight junction protein genes in mouse Brain Capillary endothelial cells highly purified by magnetic cell sorting
Journal of neurochemistry, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Yuki Katsukura, Tomoko Asashima, Tetsuya TerasakiAbstract:Tight junctions (TJs) are an important component of the blood-Brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of Brain Capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 106 cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the Brain Capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-Brain barrier in cooperation with claudin-5.
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establishing a method to isolate rat Brain Capillary endothelial cells by magnetic cell sorting and dominant mrna expression of multidrug resistance associated protein 1 and 4 in highly purified rat Brain Capillary endothelial cells
Pharmaceutical Research, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Tetsuya TerasakiAbstract:To establish a method for isolating highly purified Brain Capillary endothelial cells (BCECs) from rat Brain by using magnetic cell sorting, and clarify the expression levels of multidrug resistance-associated protein (Mrp) subtypes in these highly purified BCECs. The cells were prepared from the Capillary enriched-fraction by enzyme digestion, and reacted with anti-PECAM-1 antibody. The cell sorting was performed by autoMACS. The mRNA levels were measured by quantitative real-time PCR analysis. From five rats, 2.3 × 106 cells were isolated in the PECAM-1(+) fraction and the percentage of labeled cells in this was 85.9%. PECAM-1, claudin-5 and Tie-2 mRNA were concentrated in the PECAM-1(+) fraction compared with rat Brain. The contamination by neurons and astrocytes was markedly less than in the Brain Capillary fraction prepared by the glass bead column method. Mrp1 and 4 were predominantly expressed in the PECAM-1(+) fraction at similar levels to Mdr1a. The mRNA levels of Mrp5 and 3 were 10.6 and 7.60% of that of Mrp1, respectively. This new purification method provides BCECs with less contamination by neural cells. In the isolated BCECs, Mrp1 and 4 are predominantly expressed, suggesting that they play an important role at the rat blood-Brain barrier.
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exogenous expression of claudin 5 induces barrier properties in cultured rat Brain Capillary endothelial cells
Journal of Cellular Physiology, 2007Co-Authors: Sumio Ohtsuki, Hirofumi Yamaguchi, Tomoko Asashima, Saori Sato, Mayu Kamoi, Tetsuya TerasakiAbstract:Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-Brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat Brain Capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat Brain Capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat Brain Capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat Brain Capillary fraction (per total mRNA) was 35.6-fold greater than that in whole Brain, while the level of claudin-12 mRNA was only 13.9% of that in whole Brain, suggesting that expression of claudin-12 mRNA is not restricted to Brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell–cell boundaries. The permeability surface product of [14C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 × 10−3 and 11.6 × 10−3 cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in Brain capillaries, and plays a key role in the appearance of barrier properties of Brain Capillary endothelial cells. J. Cell. Physiol. 210: 81–86, 2007. © 2006 Wiley-Liss, Inc.