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

Shimomura, Keita; Okura, Takashi; Kato, Sayaka; Couraud, Pierre Olivier; Schermann, J; Terasaki, Tetsuya; Deguchi, Yoshiaki

doi:10.1186/2045-8118-10-8

Cited by 67 publications

(134 citation statements)

References 23 publications

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“…4). Intracellular alkalization in the presence of 30 mM NH 4 Cl markedly reduced the uptake, while intracellular acidification induced by pretreatment with and subsequent removal of NH 4 Cl stimulated the uptake, suggesting that the uptake was driven by an oppositely directed proton gradient.…”

Section: Determination Of Apomorphine R(−)-apomorphine or S(+)-apomormentioning

confidence: 97%

“…Uptake was also measured at medium pH values of 6.0, 7.4 and 8.4. To examine the influence of intracellular pH (pHi), the uptake was measured in the presence of 30 mM NH 4 Cl to elevate pHi. 4,6) To measure the uptake at acidic pHi, extracellular NH 4 Cl was removed after preincubation with 30 mM NH 4 Cl, because intracellular NH 3 rapidly diffuses out of the cells, resulting in the accumulation of protons released from NH 4 + during NH 3 generation in the cells.…”

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

“…To examine the influence of intracellular pH (pHi), the uptake was measured in the presence of 30 mM NH 4 Cl to elevate pHi. 4,6) To measure the uptake at acidic pHi, extracellular NH 4 Cl was removed after preincubation with 30 mM NH 4 Cl, because intracellular NH 3 rapidly diffuses out of the cells, resulting in the accumulation of protons released from NH 4 + during NH 3 generation in the cells. In the inhibition study, the initial uptake (30 µM for 1 min) was measured in the presence of selected organic cations, i.e., tetraethylammonium (substrate of organic cation transporters 1-3; OCT1-3), carnitine (a substrate of carnitine/ organic cation transporter 2; OCTN2), amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of the proton-coupled organic cation antiporter) at the concentration of 1 mM.…”

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

“…In vitro uptake studies were performed as previously reported. 4) hCMEC/ D3 cells were seeded on type I collagen-coated 24-well plates (BIOCOAT, Becton Dickinson, Bedford, MA, U.S.A.) at a density of 20000 cells/cm 2 . The cells reached confluence at 3-4 d after seeding, and then were washed twice with 1 mL of incubation buffer (122 mM NaCl, 3 mM KCl, 25 mM NaHCO 3 , 1.2 mM MgSO 4 , 1.4 mM CaCl 2 , 10 mM D-glucose, 10 mM HEPES, pH 7.4) and preincubated with incubation buffer for 20 min at 37°C.…”

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

“…2,3) Recently, we have reported that hCMEC/D3 cells retain activity of the proton-coupled organic cation antiporter, which is functionally expressed in rodent BBB. 4) Although the molecular entity of the protoncoupled organic cation antiporter remains unknown, this antiporter mediates blood-to-brain transport of central nervous system (CNS)-acting cationic drugs such as oxycodone and diphenhydramine, and generates a marked concentration gradient of these drugs across the BBB, with a 3 to 5 times higher unbound concentration in the brain.…”

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Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3

Okura

Higuchi

Kitamura

et al. 2014

Biological & Pharmaceutical Bulletin

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R( )-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson's disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R( )-apomorphine and its S-enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R( )-or S( )-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy-and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R( )-and S( )-apomorphine increased with time. The initial uptake velocities of R( )-and S( )-apomorphine were concentration-dependent, with similar K m and V max values. The cell-to-medium (C/M) ratio of R( )-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R( )-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R( )-apomorphine transport in human blood-brain barrier (BBB) model cells is similar to S( )-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium-or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.Key words apomorphine; blood-brain barrier; proton-coupled organic cation antiporter; hCMEC/D3 cell R(−)-Apomorphine, a dopamine agonist, has been approved as a subcutaneous injection formulation for rescue management of motor function impairment associated with levodopa therapy in patients with Parkinson's disease. It has been reported to have a 12 times higher unbound concentration in the brain than the blood (brain-to-blood unbound concentration ratio, K puu =12, 1) whereas S(+)-apomorphine, the dopamine receptor-inactive enantiomer, has a K puu of 5.1) These results suggest that both enantiomers are actively transported into the brain across the blood-brain barrier (BBB) in rats. However, the mechanism of blood-to-brain transport of this drug across the BBB remains unknown.The immortalized human brain capillary endothelial cell line, hCMEC/D3 has been extensively validated as a BBB model by means of pharmacological, toxicological, immunological and infection studies in numerous laboratories worldwide, and it is established that hCMEC/D3 cells retain many of the morphologi...

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Section: Determination Of Apomorphine R(−)-apomorphine or S(+)-apomormentioning

confidence: 97%

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

Section: Chemicals R(−)-apomorphine and S(+)-apomorphinementioning

confidence: 99%

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confidence: 99%

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Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3

Okura

Higuchi

Kitamura

et al. 2014

Biological & Pharmaceutical Bulletin

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The ergothioneine transporter (ETT): substrates and locations, an inventory

2022

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In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell‐surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well‐defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.

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Involvement of a proton‐coupled organic cation antiporter in the blood–brain barrier transport of amantadine

Suzuki

Aoyama

Suzuki

et al. 2016

Biopharm & Drug Disp

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The blood-to-brain transport of amantadine, a weak N-methyl-d-aspartate (NMDA) antagonist, has been shown previously to participate in the cationic drug-sensitive transport system across the mouse blood-brain barrier (BBB). The purpose of the present study was to characterize the influx transport system by means of both an in situ mouse brain perfusion technique and in vitro studies using rat immortalized brain capillary endothelial cells (GPNT). The observed concentration-dependent initial uptake rate of [(3) H]amantadine suggested the involvement of a carrier-mediated transport mechanism. The normal uptake at physiological pH 7.4 was decreased by 72.9% in acidic perfusate, while it was increased by 35.3% in alkaline perfusate. These results suggest that pH-dependent transport is regulated by utilizing an oppositely directed proton gradient as a driving force. In addition, the [(3) H]amantadine uptake was moderately inhibited by the adamantane structural analogs (rimantadine and memantine) and other cationic drugs (pyrilamine, clonidine, nicotine, etc.), but not by substrates or inhibitors of the well-characterized organic cation transporters (tetraethylammonium, l-carnitine and choline). A similar inhibition pattern was observed between the in vivo studies and the in vitro experiments. These results indicate that the influx transport for amantadine across the BBB involves a proton-coupled organic cation antiporter. Copyright © 2016 John Wiley& Sons, Ltd.

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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

Cited by 67 publications

References 23 publications

Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3

Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3

The ergothioneine transporter (ETT): substrates and locations, an inventory

Involvement of a proton‐coupled organic cation antiporter in the blood–brain barrier transport of amantadine

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