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Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3
2014 Volume 37 Issue 2 Pages 286-291
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2014 Volume 37 Issue 2 Pages 286-291
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 Km and Vmax 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.
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, Kpuu=12,1) whereas S(+)-apomorphine, the dopamine receptor-inactive enantiomer, has a Kpuu 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 morphological and functional characteristics of human brain capillary endothelial cells.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 proton-coupled 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.4–7)
R(−)-Apomorphine and S(+)-apomorphine each have a tertiary amine moiety, which is cationized at physiological pH, as is also the case in oxycodone and diphenhydramine. Thus, we hypothesized that apomorphine is actively transported into the brain across the BBB by the proton-coupled organic cation antiporter. The aim of this study, therefore, was to test this idea by examining uptake of R(−)-apomorphine and S(+)-apomorphine by hCMEC/D3 cells, as a model of the human BBB.
R(−)-Apomorphine and S(+)-apomorphine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) and Sigma (St. Louis, MO, U.S.A.), respectively. All other chemicals and reagents were commercial products of reagent grade.
Cell CultureThe hCMEC/D3 cell line was kindly provided by Dr. Couraud (Insititut Cochin, Paris, France). The cell line was originally produced by immortalizing brain endothelial microvascular cells isolated from tissue surgically excised from the temporal lobe of an adult female with epilepsy, by means of lentiviral transduction of the cells with catalytic subunit of human telomerase and SV40-T antigen.2) hCMEC/D3 cells were cultured in EBM-2 medium (TaKaRa Bio, Shiga, Japan) supplemented with 2.5% fetal bovine serum, 0.025% vascular endothelial growth factor, 0.025% insulin-like growth factor-1, 0.025% epidermal growth factor, 5 µg/mL basic fibroblast growth factor, 0.01% hydrocortisone, 1% penicillin-streptomycin and 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) on rat collagen type I coated dishes. They were maintained in 95% air and 5% CO2 at 37°C.
In Vitro Uptake StudiesThe hCMEC/D3 cells used for the experiments were between passages 25 and 35. 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/cm2. 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 NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4) and preincubated with incubation buffer for 20 min at 37°C. After preincubation, the buffer (0.25 mL) containing R(−)- or S(+)-apomorphine (3–1000 µM) was added to initiate uptake. The cells were incubated at 37°C for a designated time (0.25, 1, 3 or 10 min), and then washed three times with 1 mL of ice-cold incubation buffer to terminate the uptake. The cells were collected and the content of R(−)- or S(+)-apomorphine was determined as described below. The protein content of collected cells after solubilization with 1 N NaOH was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce Chemical Co., Rockford, IL, U.S.A.).
Uptake was expressed as the cell-to-medium ratio (µL/mg protein) obtained by dividing the uptake amount by the concentration of R(−)- or S(+)-apomorphine in the incubation buffer. In order to estimate the kinetic parameters, R(−)- or S(+)-apomorphine uptake (3, 10, 30, 100, 300 and 1000 µM, for 1 min) was measured as the initial uptake velocity (nmol/mg protein/min) and it was fitted to the following equation by means of nonlinear least-squares regression analysis with Prism software (Graphpad, San Diego, CA, U.S.A.):
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where v, s, Vmax, Km and Kns represent the initial uptake velocity (nmol/mg protein/min), R(−)- or S(+)-apomorphine concentration (µM), maximum uptake velocity (nmol/mg protein/min), Michaelis constant (µM), and the first-order constant for the non-saturable component (µL/mg protein/min), respectively.
The initial uptake of R(−)-apomorphine (30 µM for 1 min) was also measured under ATP-depleted (pretreatment with 0.1% sodium azide for 20 min), Na+-free (Na+ was replaced with N-methylglucamine) and membrane potential-disrupted (Na+ was replaced with K+) conditions. 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 NH4Cl to elevate pHi.4,6) To measure the uptake at acidic pHi, extracellular NH4Cl was removed after preincubation with 30 mM NH4Cl, because intracellular NH3 rapidly diffuses out of the cells, resulting in the accumulation of protons released from NH4+ during NH3 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. The R(−)-apomorphine uptake (10–1000 µM, for 1 min) was measured in the absence and presence of 100 µM diphenhydramine.
Determination of ApomorphineR(−)-Apomorphine or S(+)-apomorphine was determined by ultra-high-performance liquid chromatography (UPLC) with a fluorescence detector. The collected cells were dispersed in 0.1% ethylenediaminetetraacetic acid (EDTA) and 0.15% ascorbic acid solution containing 100 µM bupropion as an internal standard and acetonitrile (3 volumes) was added. The mixture was shaken on a horizontal shaker for 2 min and then centrifuged for 3 min at 12000 rpm. An aliquot of the supernatant was injected into the Nexera® UPLC system (Shimadzu, Kyoto, Japan), which consisted of a pump (LC-30AD), autosampler (SIL-30AC), column oven (CTO-20AC), UV detector (SPD-20A), fluorescence detector (RF-20Axs) and system controller (CBM-20A). The analytical column used was a Shim-pack XR-ODS III (2.0 mm ×50 mm, 1.6 µm particle size, Shimadzu). The UPLC separation was carried out at a flow rate of 0.4 mL/min with a mobile phase containing 25% acetonitrile and 10 mM phosphate buffer. Fluorescence detection of apomorphine was done at excitation and emission wavelengths of 272 and 459 nm, respectively. UV detection of bupropion was performed at 205 nm. The retention times of apomorphine and bupropion were 0.9 and 3.0 min, respectively. The detection limit for quantification of apomorphine was 0.1 pmol.
Statistical AnalysisStatistical analysis was performed by using Student’s t-test and one-way analysis of variance followed by Dunnett’s test for single and multiple comparisons, respectively. Differences were considered statistically significant at p<0.05.
The uptakes of both R(−)- and S(+)-apomorphine into hCMEC/D3 cells increased with time, and reached plateau levels at 3 min (Fig. 1). The value of the cell-to-medium (C/M) ratio for R(−)-apomorphine at each time was similar to that for S(+)-apomorphine. The initial uptake velocities of R(−)- and S(+)-apomorphine were concentration-dependent (Fig. 2). Eadie–Hofstee plots for R(−)- and S(+)-apomorphine uptake each gave a single straight line, indicating involvement of a single saturable process. Kinetic analysis provided Km values of 42 and 55 µM, Vmax values of 9.8 and 13 nmol/mg protein/min, Vmax/Km values of 232 and 236 µL/mg protein/min and Kns values of 14.7 and 12.6 µL/mg protein/min for R(−)- and S(+)-apomorphine, respectively. The saturable transport of R(−)- and S(+)-apomorphine estimated from the kinetic parameters accounted for more than 90% of total transport at the concentration of 30 µM. The R(−)-apomorphine uptake was assessed at the concentration of 30 µM, in the subsequent ATP depletion, ion-dependence and inhibition studies.
Uptake of R(−)-apomorphine (●) or S(+)-apomorphine (○) (30 µM each) was measured at 37°C. Each point represents the mean±S.E. of four determinations.
Uptake of R(−)-apomorphine or S(+)-apomorphine (3, 10, 30, 100, 300 and 1000 µM, for 1 min) was measured at 37°C. Each point represents the mean±S.E. of four determinations. The solid curve, dotted line and curve represent estimated total, nonsaturable and saturable uptakes, respectively (A, B). V and S represent initial uptake velocity (nmol/mg protein/min) of the saturable component and concentration of R(−)- or S(+)-apomorphine (μM), respectively (C, D).
The C/M ratio was significantly inhibited by pretreatment with sodium azide (NaN3), but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium ion (Fig. 3), suggesting that the uptake was dependent on metabolic energy, but was sodium ion- and membrane potential-independent. The C/M ratio was decreased at acidic extracellular pH (6.0), whereas alkaline extracellular pH (8.4) did not cause significant increase in the C/M ratio (Fig. 4). Intracellular alkalization in the presence of 30 mM NH4Cl markedly reduced the uptake, while intracellular acidification induced by pretreatment with and subsequent removal of NH4Cl stimulated the uptake, suggesting that the uptake was driven by an oppositely directed proton gradient.
R(−)-Apomorphine uptake (30 µM, for 1 min) was measured in the absence (control) or presence of 0.1% sodium azide (NaN3) (A). Sodium azide was preincubated for 20 min. R(−)-Apomorphine uptake (30 µM, for 1 min) was measured in sodium ion-containing buffer (control) or buffer in which sodium ion had been replaced with N-methylglucamine (NMG) or potassium to disrupt membrane potential (KCl). Each column represents the mean±S.E. of four determinations. Asterisks show a significant difference, *** p<0.001 vs. control.
R(−)-Apomorphine uptake (30 µM, for 1 min) was measured in buffer at pH values of 6.0, 7.4 and 8.4 (A). R(−)-Apomorphine uptake (30 µM, for 1 min) was measured under conditions of intracellular alkalization (pHi↑) and acidification (pHi↓) induced by NH4Cl treatment (B). Each column represents the mean±S.E. of four determinations. Asterisks show a significant difference, ** p<0.01 and *** p<0.001 vs. pH 7.4 or control.
The C/M ratio for R(−)-apomorphine was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine at the concentration of 1 mM, while tetraethylammonium (a substrate of OCTs) and carnitine (a substrate of OCTN2) did not affect the uptake (Fig. 5). Lineweaver–Burk plots of R(−)-apomorphine uptake in the presence and absence of diphenhydramine (100 µM) intersected at the ordinate axis, indicating that diphenhydramine competitively inhibited R(−)-apomorphine (Fig. 6).
R(−)-Apomorphine uptake (30 µM, for 1 min) was measured in the absence (control) or presence of tetraethylammonium (TEA), carnitine, amantadine, verapamil, pyrilamine or diphenhydramine at the concentration of 1 mM. Each column represents the mean±S.E. of four determinations. Asterisks show a significant difference, *** p<0.001 vs. control.
R(−)-Apomorphine uptake (10–1000 µM, for 1 min) was measured in the absence (○) or presence of 100 µM diphenhydramine (●). Each point represents the mean±S.E. of three determinations.
In the current study, the role of the proton-coupled organic cation antiporter in R(−)-apomorphine transport at the human BBB was examined by using human brain endothelial cell line hCMEC/D3 as a model. The time course and kinetic parameters of R(−)-apomorphine uptake into hCMEC/D3 cells were similar to those of the S-enantiomer, and the transport of R(−)-apomorphine was dependent on an oppositely directed proton gradient.
The uptakes of R(−)- and S(+)-apomorphine were time- and concentration-dependent (Figs. 1, 2). Calculated uptake clearances (Vmax/Km) for R(−)- and S(+)-apomorphine (232 and 236 µL/min/mg protein, respectively) were in good agreement with those of diphenhydramine (220 µL/min/mg protein) and [3H]pyrilamine (184 µL/min/mg protein) in hCMEC/D3 cells,4) and the values were 2 orders of magnitude greater than that for OCTN2-mediated transport of L-carnitine (0.14 µL/min/mg protein) in hCMEC/D3 cells.8) R(−)-Apomorphine uptake was significantly inhibited by pretreatment with metabolic inhibitor, but was insensitive to extracellular sodium ion and to membrane potential (Fig. 3). Furthermore, R(−)-apomorphine uptake by hCMEC/D3 cells showed pH-dependency characteristic of a proton-coupled antiporter (Fig. 4). These results suggest that R(−)-apomorphine transport is mediated by the previously reported proton-coupled organic cation antiporter, which is an organic cation transport system different from OCTs and OCTNs.6,9–11) The results of inhibition study (Figs. 5, 6) also support the idea that the proton-coupled organic cation antiporter is involved in the transport of R(−)-apomorphine in hCMEC/D3 cells, because the R(−)-apomorphine uptake was significantly decreased by substrates or inhibitors of this antiporter (amantadine, verapamil, pyrilamine and diphenhydramine), but not by a substrate of OCTs (tetraethylammonium) or a substrate of OCTN2 (carnitine).
Kpuu values are the ratio of blood-to-brain influx clearance/brain-to-blood efflux clearance (CLinflux/CLefflux), and thus Kpuu values greater than unity suggest active transport in the direction of blood to brain across the BBB. The proton-coupled organic cation transporter appears to show no marked species difference, at least between rat and human brain endothelial cells (TR-BBB13 and hCMEC/D3 cells, respectively).4) Further, the uptake clearances in TR-BBB13 and hCMEC/D3 cells for diphenhydramine and pyrilamine are in reasonable agreement with in vivo CLinflux values determined by the rat in situ perfusion method and human PET study with [11C]pyrilamine.4,12) Thus, the proton-coupled organic cation antiporter-mediated transport of R(−)-apomorphine with relatively high uptake clearance in hCMEC/D3 cells suggests the occurrence of efficient blood-to-brain transport of R(−)-apomorphine across the human BBB. The proton-coupled organic cation antiporter may play an important role in the brain distribution of R(−)-apomorphine after rescue dosing in patients with Parkinson’s disease, and consequently may be a determinant of therapeutic efficacy.
Several drugs have been reported to be proton-coupled organic cation antiporter substrates, including pramipexole,13) oxycodone,6) pyrilamine,4,6) 3,4-methylenedioxymethamphetamine,9) clonidine,10) diphenhydramine,4,7,14) nicotine,15,16) and propranolol.11) These substrate drugs, like apomorphine, have a secondary or tertiary amine moiety and a hydrophobic group in their structures. Thus, a basic amine structure with a hydrophobic group may be required for recognition as a substrate by the proton-coupled organic cation antiporter. The wide variety of chemical structures of these substrate drugs indicates poly-specificity of this antiporter. Sam et al. reported that the Kpuu value for R(−)-apomorphine (12) is higher than that for S(+)-apomorphine (5).1) Our findings of similar uptake time courses and kinetic parameters for R(−)- and S(+)-apomorphine suggest that the higher Kpuu value of R(−)-apomorphine than that of S(+)-apomorphine is not due to a difference of CLinflux values. To clarify the mechanism underlying the different Kpuu values, further studies are needed, including measurement of CLefflux values of R(−)- and S(+)-apomorphine.
Apomorphine has tertiary amine and phenolic hydroxyl with the reported pKa values of 8.83 and 7.42 at 37°C.17) According to the pKa values, major formations of apomorphine should be cation at pH 6.0, cation or zwitterion at pH 7.4 and zwitterion or anion at pH 8.4, whereas slight uncharged formation should be existed at any range of pH values. Thus, the simple diffusion process according to the pH-partition theory could not be solely responsible for the apomorphine uptake. Indeed, it was estimated from the kinetic parameters that the non-saturable component of apomorphine uptake (30 µM) at pH 7.4 was less than 10% of the total uptake in hCMEC/D3 cells. The extracellular alkalization (pH 8.4) did not cause significant increase in the apomorphine uptake although the intracellular acidification stimulated the uptake (Fig. 4). Assuming that the proton-coupled organic cation antiporter can accept only cationic or zwitterionic formation of apomorphine, the extracellular alkalization decreases the acceptable formations of apomorphine, though the alkalization increases the driving force of the proton-coupled organic cation antiporter. This may be one of the reasons for the no significant increase in the apomorphine uptake by the extracellular alkalization (pH 8.4).
In conclusion, the time course and kinetic parameters for R(−)-apomorphine uptake were similar to those of the S-enantiomer in human BBB model cells. The transport of R(−)-apomorphine was dependent on an oppositely directed proton gradient, but was sodium ion- and membrane potential-independent, in accordance with the reported properties of the proton-coupled organic cation antiporter. Our results indicate that proton-coupled organic cation antiporter-mediated transport of R(−)-apomorphine at the BBB plays a role in determining the therapeutic efficacy of rescue dosing of this drug.
We thank Dr. Pierre-Olivier Couraud (Institut Cochin, Paris, France) for supplying hCMEC/D3 cells under license from INSERM. This work was supported in part by a Grant-in-Aid for Scientific Research provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
