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Interaction between l -DOPA and 3-O-methyl-l -DOPA for transport in immortalised rat capillary cerebral endothelial cells
Neuropharmacology 38 (1999) 1371 – 1380 www.elsevier.com/locate/neuropharm
Interaction between L-DOPA and 3-O-methyl-L-DOPA for transport in immortalised rat capillary cerebral endothelial cells P. Gomes, P. Soares-da-Silva * Institute of Pharmacology & Therapeutics, Faculty of Medicine, 4200 Porto, Portugal Accepted 22 February 1999
Abstract The present study aimed to determine the kinetics of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of interaction with 3-O-methyl-LDOPA (3-OM-L-DOPA), sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC), N-(methylamino)-isobutyric acid (MeAIB) and sodium. Non-linear analysis of the saturation curves for L-DOPA and 3-OM-L-DOPA revealed in RBE 4 cells Km values (in mM) of 72 (53, 91) and 40 (25, 57) and in RBE 4B cells Km values (in mM) of 60 (46, 74) and 44 (13, 75), respectively. IC50 values for 3-OM-L-DOPA (RBE 4, 642 [542, 759] mM; RBE 4B, 482 [475, 489] mM) obtained in the presence of a nearly saturating (250 mM) concentration of L-DOPA were greater than the corresponding Ki values (RBE 4, 143 [121, 170] mM; RBE 4B, 93 [92, 95] mM) obtained in the presence of a nearly saturating (250 mM) concentration of 3-OM-L-DOPA; this is compatible with a competitive type of interaction between L-DOPA and 3-OM-L-DOPA. Uptake of both L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells was sensitive to BHC with similar IC50 values. MeAIB (up to 2.5 mM) was found not to interfere with the uptake of both L-DOPA and 3-OM-L-DOPA. Uptake of (250 mM) L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20 – 140 mM). Homogenates of both cell lines were endowed with considerable COMT activity. Incubation of RBE 4 and RBE 4B cells with L-DOPA (25 mM) in the presence of a methyl donor (S-adenosyl-L-methionine) resulted in the formation of 3-OM-L-DOPA; this was abolished by 1 mM tolcapone. The fractional outflow of intracellular L-DOPA through the luminal and abluminal cell side was not affected by the presence of intracellular 3-OM-L-DOPA. The fractional outflow of exogenous 3-OM-L-DOPA applied from the luminal cell border was similar to that observed for 3-OM-L-DOPA with origin in L-DOPA. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and 3-OM-L-DOPA behaves as a competitive inhibitor for the uptake of L-DOPA. This, however, only occurs for luminal cell inward movement but not for abluminal cell outward movement of the substrates. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Brain capillary endothelial cells; L-DOPA; 3-O-Methyl-L-DOPA; Cellular transport
1. Introduction Replenishment therapy in Parkinson’s disease with (L-DOPA), a direct precursor amino acid of dopamine, has been in the past decades the ultimate strategy to activate the failing dopaminergic system in the parkinsonian brain. In advanced stages of the disease, the usefulness of L-DOPA therapy is limited by a variety of motor complications L-3,4-dihydroxyphenylalanine
* Corresponding author. Tel.: + 351-2-595694; fax: + 351-25502402. E-mail address: [email protected] (P. Soares-da-Silva)
that include: peak-dose dyskinesia; end-of-dose or ‘wearing off’ phenomenon; dystonia; ‘on-off’ phenomenon; and dyskinesia. Some of these motor complications, namely the ‘wearing off’ phenomenon have been suggested to be related to kinetic factors that affect the absolute and relative plasma concentrations of L-DOPA and 3-O-methyl-L-DOPA (3-OM-L-DOPA) (Furgeson et al., 1976; Rivera-Calimlim et al., 1977; Nutt and Fellman, 1984). Although the decarboxylation of L-DOPA is believed to represent a major pathway of its metabolism, methylation into 3-OM-L-DOPA by catechol-O-methyltransferase (COMT) assumes particular relevance in
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conditions of AAAD inhibition (Sharpless et al., 1973; Ma¨nnisto¨ and Kaakkola, 1989). There is, however, some controversy over the extent to which circulating 3-OM-L-DOPA may interfere with the therapeutic response to L-DOPA in parkinsonian patients. On one hand, patients with L-DOPA-induced dyskinesias have been reported to present higher plasma concentrations of 3-OM-L-DOPA (Reilly et al., 1980), systemic 3-OML-DOPA decreased brain uptake of [14C]-L-DOPA (Gervas et al., 1983) or reduced response to L-DOPA (Reches and Fahn, 1982; Reches et al., 1982), and COMT inhibitors potentiate the effects of L-DOPA (Linden et al., 1988; Himori and Mishima, 1994). On the other hand, plasma 3-OM-L-DOPA concentrations in parkinsonian patients treated with L-DOPA on a long-term basis reflect daily L-DOPA dosage and do not vary markedly during the day (Nutt et al., 1987). These observations, plus the fact that 3-OM-L-DOPA makes a small contribution to the total concentration of large neutral amino acids (LNAAs) competing with L-DOPA for transport at the blood-brain barrier (Nutt et al., 1987), support the contention that circulating 3-OM-L-DOPA may not be an important determinant of the clinical response to L-DOPA. In agreement with this view is the observation that infusion of 3-OM-LDOPA to produce plasma concentrations (39 mmol/l) equivalent to those seen in patients on chronic L-DOPA therapy failed to alter the blood-brain transfer rate of 6-[18F]-fluoroDOPA (Guttman et al., 1992). Under physiological conditions LNAAs compete with each other for transport into the brain (Pardridge and Oldendorf, 1977; Pardridge, 1995), and the availability of L-DOPA to brain tissue can be compromised by the presence of high levels of LNAAs in plasma (Alexander et al., 1994). LNNAs and L-DOPA are believed to share the same transporters to cross the BBB (Frankel et al., 1989; Nutt et al., 1989; Woodward et al., 1993; Alexander et al., 1994). LNAAs are transported at the level of the BBB by the L-type amino acid transporter, which is sodium-independent and sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC). The A-type amino acid transporter has also been shown to transport phenylalanine, but this transporter is mainly involved in the transfer of small nonessential amino acids such as alanine and glycine. Differentiation between A- and L-type is based on sodium-dependence and sensitivity to N-(methylamino)-isobutyric acid (MeAIB) and insensitivity to BHC. The ASC-type of amino acid transporter is a sodium dependent transporter and insensitive to both BHC and MeAIB (Wade and Katzman, 1975a,b; Audus and Borchardt, 1986; Sanchez del Pino et al., 1992). The aim of the present study was to define the kinetics of L-DOPA uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4
and RBE 4B) and the type of interaction produced by 3-OM-L-DOPA. This cell line was obtained by transfection of rat brain microvessel endothelial cells with a plasmid containing the E1A adenovirus gene. These cells display a non-transformed endothelial phenotype expressing the brain microvessel-associated enzymes— g-glutamyl-transpeptidase, alkaline phosphatase and Pglycoprotein (Roux et al., 1994; Begley et al., 1996; El Hafny et al., 1997). We have also attempted to characterise the L-DOPA transporter in these cells, namely its sodium dependence and its sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC) and N(methylamino)-isobutyric acid (MeAIB).
2. Methods
2.1. Cell culture RBE 4 and RBE 4B clones were kindly supplied by Dr Franc¸oise Roux (INSERM U. 26, Hoˆpital Fernand Widal, Paris, France) and maintained in a humidified atmosphere of 5% CO2, 95% air at 37°C. RBE 4 (passages 25–29) and RBE 4B cells (passages 2– 6) were grown in Minimum Essential Medium/Ham’s F10 (1:1) (Sigma Chemical Co, MO, USA) supplemented with 300 ng/ml neomycine, 10% foetal bovine serum (Sigma), 1 ng/ml basic fibroblast growth factor, 100 U/ml penicillin G, 0.25 mg/ml amphotericin B, 100 mg/ml streptomycin (Sigma) and 25 mM N-2-hydroxyethylpiperazine-N%-2-ethanosulfonic acid (HEPES; Sigma). The cell medium was changed every 2 days, and the cells reached confluence after 3–4 days of incubation. For subculturing, the cells were dissociated with 0.05% trypsin-EDTA (Sigma), diluted 1:7 and subcultured in petri dishes with a 21-cm2 growth area (Costar, Badhoevedorp, The Netherlands). For uptake studies, the cells were seeded in collagen-treated 24-well plastic culture clusters (internal diameter 16 mm, Costar) at a density of 40 000 cells per well (2.0×104 cells/cm2) or, depending on the experiment, onto collagen treated 0.2 mm polycarbonate filter supports (internal diameter 12 mm Transwell, Costar). For 24 h prior to each experiment, the cell medium was free of foetal bovine serum and basic fibroblast growth factor. Experiments were generally performed 2–3 days after cells reached confluence and 6 days after initial seeding and each cm2 contained about 50 mg of cell protein.
2.2. Transport studies On the day of the experiment, the growth medium was aspirated and the cells were washed with Hanks’ medium at 4°C; thereafter, the cell monolayers were preincubated for 30 min in Hank’s medium at 37°C. The Hanks’ medium had the following composition
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(mM): NaCl 137, KCl 5, MgSO4 0.8, Na2HPO4 0.33, KH2PO4 0.44, CaCl2 0.25, MgCl2 1.0, Tris – HCl 0.15 and sodium butyrate 1.0, pH 7.4. In experiments performed in the presence of different concentrations of sodium, NaCl was replaced by an equimolar concentration of choline chloride. The incubation medium also contained benserazide (50 mM), pargyline (100 mM) and tolcapone (1 mM) in order to inhibit the enzymes aromatic L-amino acid decarboxylase, monoamine oxidase and catechol-O-methyltransferase, respectively. During preincubation and incubation, the cells were continuously shaken and maintained at 37°C. Apical uptake was initiated by the addition of 2 ml Hanks’ medium with a given concentration of the substrate (L-DOPA or 3-OM-L-DOPA). Time course studies were performed in experiments in which cells were incubated with 1 mM substrate for 1, 3, 6 and 12 min. Saturation experiments were performed in cells incubated for 6 min with increasing concentrations of L-DOPA (10 – 1000 mM). In experiments designed to study the effects of 3-OM-LDOPA, BHC, and MeAIB upon the uptake of LDOPA, RBE 4 and RBE 4B cells were preincubated for 30 min in the presence of the compounds to be tested. After preincubation, cells were incubated for 6 min in Hanks’ medium with 250 mM L-DOPA. Uptake was terminated by the rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette followed by a rapid wash with cold Hanks’ medium and the addition of 250 ml of 0.2 mM perchloric acid. The acidified samples were stored at 4°C before injection into the high pressure liquid chromatograph for the assay of L-DOPA or 3-OM-LDOPA. In another series of experiments, cells were cultured in polycarbonate supports, the substrates being applied from the apical side of the monolayer. The incubation medium used in this series of experiments was similar to that described above, in some experiments, the medium contained tolcapone (1 mM) in order to inhibit COMT. The upper and lower chambers contained 400 and 1000 ml, respectively. Cells were pre-incubated for 30 min and, thereafter, incubated in the presence of L-DOPA or 3-OM-L-DOPA. [14C]-Sorbitol (0.4 mM) was used to estimate paracellular fluxes and extracellular trapping of substrates. At the end of the incubation, cells were placed on ice and the medium bathing the apical and basal cell borders was collected, acidified with 2 M perchloric acid and stored at 4°C until it was assayed for L-DOPA and 3-OM-L-DOPA. The cells were washed with ice-cold Hanks’ medium and added with 0.2 mM perchloric acid (200 and 1000 ml in the upper and lower chambers, respectively); the acidified samples were stored at 4°C before injection into the high pressure liquid chromatograph for the assay of L-DOPA and 3-OM-L-DOPA.
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2.3. Assay of L -DOPA and 3 -OM-L -DOPA L-DOPA and 3-OM-L-DOPA were quantified by means of high pressure liquid chromatography with electrochemical detection, as previously reported. The high-pressure liquid chromatograph system consisted of a pump (Gilson model 302; Gilson Medical Electronics, Villiers le Bel, France) connected to a manometric module (Gilson model 802 C) and a stainless-steel 5-mm ODS column (Biophase; Bioanalytical Systems, West Lafayette, IN) of 25 cm length; samples were injected by means of an automatic sample injector (Gilson model 231) connected to a Gilson dilutor (model 401). The mobile phase was a degassed solution of citric acid (0.1 mM), sodium octylsulphate (0.5 mM), sodium acetate (0.1 M), EDTA (0.17 mM), dibutylamine (1 mM) and methanol (8% v/v), adjusted to pH 3.5 with perchloric acid (2 M) and pumped at a rate of 1.0 ml/min. Detection was carried out electrochemically with a glassy carbon electrode, an Ag/AgCl reference electrode and an amperometric detector (Gilson model 141); the detector cell was operated at 0.75 V. The current produced was monitored using the Gilson 712 HPLC software. The lower limits for detection of LDOPA and 3-OM-L-DOPA ranged from 350 to 500 fmol.
2.4. Protein assay The protein content of monolayers of OK cells was determined by the method of Bradford (Bradford, 1976), with human serum albumin as a standard.
2.5. Cell water content Cell water content was simultaneously measured in parallel experiments using [14C]inulin as an extracellular marker and tritiated water as a total water marker. Intracellular water, obtained by subtracting extracellular water from total water, was expressed as ml of cell water per mg protein. Subsequently, the cells were solubilised by 0.1% v/v Triton X-100 (dissolved in 5 mM Tris–HCl, pH 7.4) and radioactivity was measured by liquid scintillation counting.
2.6. Cell 6iability Cells were preincubated for 30 min (in the presence of 3-OM-L-DOPA, BHC or MeAIB) at 37°C and then incubated in the absence or the presence of L-DOPA for a further 6 min. Subsequently the cells were incubated at 37°C for 2 min with trypan blue (0.2% w/v) in phosphate buffer. Incubation was stopped by rinsing the cells twice with Hanks’ medium and the cells were examined using a Leica microscope. Under these conditions, more than 95% of the cells excluded the dye.
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2.7. Data analysis Km and Vmax values for the uptake of L-DOPA and 3-OM-L-DOPA, as determined in saturation experiments, were calculated by non-linear regression analysis, using the GraphPad Prism statistics software package (Motulsky et al., 1994). For the calculation of the IC50, the parameters of the equation for one site inhibition were fitted to the experimental data (Motulsky et al., 1994). Ki values were calculated as defined by Cheng and Prusoff (1973) for competitive inhibition. Fractional outflow was calculated using the expression L-DOPAfluid/(L-DOPAfluid + L-DOPAcell) where L-DOPAfluid indicates the amount of L-DOPA (in nmol/mg protein) which reached the apical or the basal chamber and L-DOPAcell (in nmol/mg protein) indicates the amount of L-DOPA accumulated in the cell monolayer. Fractional outflow of 3-OM-L-DOPA was calculated in the same manner as for L-DOPA, with the exception that it was possible to measure 3-OM-LDOPA present in the medium bathing both the apical and basal cell borders. Arithmetic means are given with S.E.M. or geometric means with 95% confidence values. Statistical analysis was done with a one-way analysis of variance (ANOVA) followed by Newman – Keuls test for multiple comparisons. A P-value less than 0.05 was assumed to denote a significant difference.
2.8. Drugs Drugs used were: 2-aminobicyclo(2,2,1)-heptane-2carboxylic acid (BHC; Sigma Chemical Company, St. Louis, MO, USA), L-b-3,4-dihydroxyphenylalanine (Sigma), 3-O-methyl-L-b-3,4-dihydroxyphenylalanine (Sigma), N-(methylamino)-isobutyric acid (MeAIB; Sigma), pargyline hydrochloride (Sigma) and tolcapone (kindly donated by late Professor Mose´ Da Prada, Hoffman La Roche, Basle, Switzerland). 3. Results To determine the rate constant of uptake, cells were incubated with 1 mM L-DOPA or 1 mM 3-OM-L-DOPA for 1, 3, 6 and 12 min (Fig. 1). The accumulation of L-DOPA and 3-OM-L-DOPA increased linearly with time for several minutes. The intracellular L-DOPA and 3-OM-L-DOPA concentration at initial rate of uptake (6 min) was several times larger that which could be expected by passive equilibration of the substrate. In fact, at 6 min incubation, mean intracellular concentration of L-DOPA and 3-OM-L-DOPA (see Table 1) was 40 – 77 times higher than the substrate concentration in the incubation medium (1 mM). The intracellular water content of cell monolayers was 7.090.7 ml/mg protein (n= 5).
Fig. 1. Time course of L-DOPA (closed squares) and 3-OM-L-DOPA (open squares) accumulation in RBE 4 and RBE 4B cells. Cells were incubated at 37°C with 1 mM of either L-DOPA or 3-OM-L-DOPA. The results reflect levels (in pmol/mg protein) of L-DOPA or 3-OM-LDOPA accumulated. Each point represents the mean of four experiments per group; vertical lines show S.E.M.
Thus, in all subsequent experiments designed to determine the kinetic parameters of L-DOPA and 3-OML-DOPA uptake, the cells were incubated for 6 min with increasing concentrations (10–1000 mM) of the substrate. The accumulation of both L-DOPA and 3OM-L-DOPA was found to be dependent on the concentration used and to be saturable at nearly 250 mM (Fig. 2). It is interesting to observe that the accumulation of L-DOPA in RBE 4B cells was twice that for 3-OM-L-DOPA in this cell line and twice that for L-DOPA and 3-OM-L-DOPA in RBE 4 cells. Km and Table 1 Mean intracellular concentrations (mmol/l) of L-DOPA and 3-OM-LDOPA in cultured RBE 4 and RBE 4B cells at 6 min incubation in the presence of non-saturating concentrations (1 mM) of the substrate
RBE 4 cells RBE 4B cells
L-DOPA (mmol/l)
3-OM-L-DOPA (mmol/l)
39.5 90.9 71.0 91.6
76.3 9 0.4 69.8 90.2
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Fig. 2. Accumulation of L-DOPA (closed squares) and 3-OM-LDOPA (open squares) in RBE 4 and RBE 4B cells. Cells were incubated for 6 min at 37°C and increasing concentrations (10 – 1000 mM) of the substrate were applied from the apical border. The results reflect levels (in pmol/mg protein/6 min) of accumulated L-DOPA or 3-OM-L-DOPA. Each point represents the mean of four experiments per group; vertical lines show S.E.M.
Vmax values for L-DOPA and 3-OM-L-DOPA uptake in both RBE 4 and RBE 4B cells are given in Table 2. Accumulated L-DOPA and 3-OM-L-DOPA were found not to be decarboxylated to their corresponding amines, dopamine and 3-methoxytyramine. Table 2 Km and Vmax values for RBE 4 and RBE 4B cells
RBE 4 cells L-DOPA 3-OM-L-DOPA RBE 4B cells L-DOPA 3-OM-L-DOPA
L-DOPA
and 3-OM-L-DOPA uptake in
Km (mM)
Vmax (nmol/mg protein/6 min)
72 (53, 91) 40 (25, 56)
15.99 0.4 15.7 9 0.5
59 (46, 74) 44 (13, 75)
23.69 0.5 12.79 0.7
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Fig. 3. Inhibition curve of L-DOPA uptake by 3-OM-L-DOPA in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 3 shows inhibition curves for 3-OM-L-DOPA obtained in the presence of a nearly saturating (250 mM) concentration of L-DOPA in RBE 4 and RBE 4B cells. As shown in Fig. 3, 3-OM-L-DOPA was found to produce a concentration dependent inhibition of LDOPA uptake with similar magnitude in both cell lines, although Ki values for 3-OM-L-DOPA in RBE 4B cells were significantly different (PB 0.05) from those in RBE 4 cells (Table 3).
Table 3 IC50 and Ki values for inhibition of L-DOPA uptake by 3-OM-LDOPA, determined in the presence of saturating (250 mM) concentrations of the substrate in cultured RBE 4 and RBE 4B cells Inhibitor
IC50 (mM)
Ki (mM)
RBE 4 cells 3-OM-L-DOPA
642 (542, 759)
143 (121, 170)
RBE 4B cells 3-OM-L-DOPA
482 (475, 489)
93 (92, 95)
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Fig. 4. Inhibition curve of L-DOPA uptake by BHC (open symbols) and MeAIB (closed symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 4 shows the effect of BHC and MeAIB on the uptake of a nearly saturating concentration (250 mM) of L-DOPA. As can be observed, BHC exerted a marked inhibitory effect upon L-DOPA uptake in both RBE 4 and RBE 4B cells, with similar Ki values (Table 4). In contrast to what was observed with BHC, MeAIB, the inhibitor of the A-type system for LNAAs Table 4 IC50 and Ki values for inhibition of L-DOPA and 3-OM-L-DOPA uptake by BHC, determined in the presence of saturating (250 mM) concentrations of the substrate in cultured RBE 4 and RBE 4B cells Substrate
IC50 (mM)
Ki (mM)
1177 (944, 1469) 1168 (846, 1614)
263 (211, 329) 161 (117, 223)
799 (678, 942) 1540 (1269, 1868)
155 (131, 182) 230 (190, 280)
RBE 4B cells L-DOPA
3-OM-L-DOPA RBE 4B cells L-DOPA
3-OM-L-DOPA
Fig. 5. Inhibition curve of 3-OM-L-DOPA uptake by BHC (open symbols) and MeAIB (closed symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
transport, was found not to change the uptake of a nearly saturating concentration of L-DOPA in either RBE 4 and RBE 4B cells (Fig. 4). The effects of BHC and MeAIB on the uptake of a nearly saturating concentration (250 mM) of 3-OM-L-DOPA were similar to those observed for L-DOPA. BHC produced a concentration dependent inhibition of 3-OM-L-DOPA accumulation in both RBE 4 and RBE 4B cells (Fig. 5), with Ki values similar to those observed for L-DOPA (Table 4). Similarly, MeAIB at concentrations up to 2.5 mM was devoid of inhibitory effect on the uptake of 3-OM-L-DOPA. To confirm the view that L-DOPA and 3-OM-LDOPA in these cell lines were taken up by the L-type amino acid transporter, another set of experiments tested the sodium dependence of the uptake of these two substrates. As shown in Fig. 6, uptake of a nearly saturating concentration (250 mM) of L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the pres-
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ence of increasing concentrations of sodium (20–140 mM). The next series of experiments explored the possibility that endogenous 3-OM-L-DOPA in these cells, i.e. formed from taken up L-DOPA, might compete with L-DOPA for outward transfer, namely at the abluminal side of the cell. For this purpose, both cell lines were first screened for COMT activity. The reaction was conducted using cell homogenates in the presence of a methyl donor and increasing concentrations of adrenaline. As can be observed in Fig. 7, both cell lines were found to O-methylate adrenaline, the amount of metanephrine formed was significantly greater (PB 0.05) in RBE 4B cells than in RBE 4 cells. (Vmax in nmol/mg protein/h: RBE 4, 3.79 0.1; RBE 4B, 4.6 9 0.1); Km values (in mM) for COMT in RBE 4 (4.2 [3.6, 4.7] mM) were similar to those in RBE 4B cells (3.6 [2.8, 4.5] mM). The next step performed consisted in the incubation of RBE 4 and RBE 4B cells cultured in
Fig. 7. O-methylation of increasing concentrations of adrenaline (0.1 to 100 mM) in homogenates of RBE 4 and RBE 4B cells. The results are levels (in nmol/mg protein/h) of metanephrine formed from added adrenaline. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 6. Effect of increasing medium concentrations of sodium (0, 20, 40, 60, 120 and 140 mM) on the uptake of L-DOPA (closed symbols) and 3-OM-L-DOPA (open symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
polycarbonate supports and the addition of L-DOPA (25 mM, a non-saturating concentration) from the luminal (apical) cell side, in the absence and the presence of tolcapone (1 mM). The parameters monitored in these experiments were the amount of L-DOPA and 3-OM-LDOPA accumulated in the cell and that appearing in the fluid bathing the luminal (only 3-OM-L-DOPA) and abluminal (L-DOPA and 3-OM-L-DOPA) cell sides. As shown in Table 5, both cell lines were able to convert L-DOPA to 3-OM-L-DOPA, though the extent of LDOPA conversion to 3-OM-L-DOPA was a very limited one (3%). Another interesting point is that outward transfer of 3-OM-L-DOPA at the apical cell side was considerably lower than at the basal cell border. In the presence of tolcapone, levels of 3-OM-LDOPA were below the detection limit of the method (9300 fmol/mg protein) and abluminal fractional outflow of L-DOPA was similar to that observed in the absence of COMT inhibition. However, the most interesting finding is that the abluminal fractional outflow of both L-DOPA and 3-OM-L-DOPA was of the same magnitude, despite the 30-fold difference in intracellular levels between the two compounds. In another series of experiments the cells were loaded from the apical cell border with low (0.5 mM) or high (25 mM) concentrations of 3-OM-L-DOPA, in order to know if abluminal fractional outflow rates might be influenced by the intracellular concentration of the substrate. As shown in Table 6, the intracellular concentration of 3-OM-LDOPA and the amount present in the fluid bathing the basal cell side depended on the concentration of substrate used, but the abluminal fractional outflow did not depend on the intracellular concentration of the substrate. It is interesting to note that while using low concentrations of substrate the intracellular levels 3-
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Table 5 Levels of L-DOPA and 3-OM-L-DOPA (in nmol/mg protein) in cell monolayers, fluid bathing the upper chamber and the lower chamber and abluminal fractional outflow (%) in RBE 4 and RBE 4B cells loaded with L-DOPA (25 mM) from the apical cell border in the absence and the presence of tolcapone (1 mM). Values are means 9 S.E.M. (n =6)a Tolcapone 1 mM
Control L-DOPA
3-OM-L-DOPA
L-DOPA
RBE 4 cells Upper chamber Cell 13.69 0.4 Lower chamber 10.4 90.2 Basal outflow 43.3 91.2
0.16 90.005 0.42 90.006 0.3290.006 43.09 0.6
12.9 9 0.3 8.5 9 0.3 39.7 9 1.1
RBE 4B cells Upper chamber Cell 13.1 90.8 Lower chamber 24.0 9 1.6 Basal outflow 64.5 9 1.5
0.13 9 0.006 0.289 0.029 0.329 0.014 53.694.0
12.8 90.9 21.2 91.0 62.5 92.3
a Levels of 3-OM-L-DOPA in experiments carried out in the presence of tolcapone were below the detection limit ( 9 300 fmol/mg protein).
OM-L-DOPA were similar to those obtained when the cells where loaded with 25 mM L-DOPA (see Table 5) and the abluminal fractional outflow is in the same order of magnitude.
4. Discussion The results presented here show that RBE 4 and RBE 4B cells transport quite efficiently L-DOPA and
Table 6 Levels of 3-OM-L-DOPA (in pmol/mg protein) in cell monolayers and fluid bathing the lower chamber and abluminal fractional outflow (%) in RBE 4 and RBE 4B cells loaded with 3-OM-L-DOPA (0.5 and 25 mM) from the luminal cell sidea 3-OM-L-DOPA 0.5 mM
25 mM
RBE 4 cells Upper chamber Cell Lower chamber Abluminal outflow
146.39 7.5 286.2911.5 66.1 91.6
5953.99 154.6 14746.4 9 900.9 71.09 1.1
RBE 4B cells Upper chamber Cell Lower chamber Abluminal outflow
281.7930.4 406.4 9 59.0 58.7 9 1.4
9268.6 9491.5 16523.49543.0 64.191.3
a
Values are means 9 S.E.M. (n= 6).
3-OM-L-DOPA through the apical (luminal) cell border and several findings demonstrate that this uptake process is a facilitated mechanism. Firstly, steady-state uptake of non-saturating concentrations of L-DOPA and 3-OM-L-DOPA showed a linear dependence on incubation time. Secondly, at an initial rate of uptake (6 min incubation) the cellular transport of L-DOPA and 3-OM-L-DOPA showed a curvilinear dependence on substrate medium concentration, suggesting that the uptake was saturable. Thirdly, 3-OM-L-DOPA and BHC markedly inhibited the uptake of L-DOPA. Evidence for the efficiency of the L-DOPA and 3-OM-LDOPA transport in RBE 4 and RBE 4B cells is the ratio of substrate concentration in cellular water to medium concentration. The intracellular L-DOPA and 3-OM-L-DOPA concentration at the initial rate of uptake (6 min) was several times (40–77) that which would be expected by passive equilibration of L-DOPA. The sensitivity of L-DOPA and 3-OM-L-DOPA uptake to BHC, but not to MeAIB, supports the view that L-DOPA and 3-OM-L-DOPA inward transfer in RBE 4 and RBE 4B cells is promoted neither by the A- nor the ASC-type of amino acid transporter, but most probably by the L-type amino acid transporter. Further evidence that fits this suggestion is that L-DOPA and 3-OM-LDOPA uptake in RBE 4 and RBE 4B cells is not dependent on sodium. The L-type, for leucine preferring, amino acid transporter is facilitative, sodium independent, and blocked by BHC (Wade and Katzman, 1975a,b; Audus and Borchardt, 1986; Sanchez del Pino et al., 1992; Pardridge, 1995). This suggestion also agrees with data obtained under in vivo experimental conditions where it is shown that L-DOPA transport across the rat BBB is markedly inhibited by BHC, but not by MeAIB (Wade and Katzman, 1975a,b). On the other hand, this may suggest that these cells lack the A-type amino acid transporter, which is also in agreement with data obtained for the BBB under in vivo experimental conditions (Wade and Katzman, 1975a,b, and references within). This would also agree with the observations of Audus and Borchardt (1986) showing that in bovine brain microvessel endothelial cell monolayers leucine uptake is markedly inhibited by L-DOPA, but not by MeAIB. Another point suggesting that L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells are transported through the L-type amino acid transporter concerns the similarity of the Km values for the uptake of both substrates and the Ki values for BHC when acting as an inhibitor for substrate uptake. In this respect, it is interesting to note that the Michaelis–Menten constant for BHC transport across the BBB (Km = 0.16 mM) under in vivo experimental conditions has been reported to be slightly lower than that for L-DOPA (Km = 0.34 mM), but similar to that
P. Gomes, P. Soares-da-Sil6a / Neuropharmacology 38 (1999) 1371–1380
for 3-OM-L-DOPA (Km =0.13 mM) (Wade and Katzman, 1975a,b). Km values for leucine uptake in bovine brain microvessel endothelial cell monolayers (Km = 0.18 mM) have also been shown (Audus and Borchardt, 1986) to be similar to those observed in RBE 4 and RBE 4B cells. On the other hand, similarities of the Km values for L-DOPA uptake in RBE 4 and RBE 4B cells and those obtained in rat renal tubular epithelial cells (135–216 mM), LLC-PK1 cells (123 mM), OK cells (14–129 mM) and Caco-2 cells (60 mM) (Soaresda-Silva et al., 1994; Vieira-Coelho and Soares-daSilva, 1997; Soares-da-Silva et al., 1998; Vieira-Coelho and Soares-da-Silva, 1998), strongly suggest that the L-DOPA transporter in brain capillary endothelial cells may be similar to that in renal and intestinal epithelial cells. The similarity of the kinetics for L-DOPA and 3OM-L-DOPA uptake and their sensitivity to BHC suggests that the inhibitory effect of 3-OM-L-DOPA on L-DOPA uptake is of the competitive type. This is also supported by the fact that IC50 values were markedly greater than Ki values, as suggested by Cheng and Prusoff (1973) for competitive inhibition. 3-OM-L-DOPA was found to produce marked inhibition of L-DOPA uptake, in agreement with previous suggestions that this metabolite of L-DOPA may limit the access of the parent compound to the brain, as was found to occur in rat brain (Wade and Katzman, 1975a,b). According to some authors (Nutt et al., 1987; Guttman et al., 1992), this may not be a major limitation for the access of L-DOPA to the brain, since steady-state plasma levels of 3-OM-L-DOPA (39 mmol/ l) in parkinsonian patients treated with L-DOPA make a small contribution to the total concentration of LNAAs competing with L-DOPA for transport at the blood–brain barrier. It is interesting, however, to observe that this concentration of circulating 3-OM-LDOPA is similar to the half saturating concentration in RBE 4 and RBE 4B cells, but below the Ki value for inhibition of L-DOPA uptake. In isolated rat renal tubules (Soares-da-Silva et al., 1994), Ki values for inhibition of L-DOPA uptake by 3-OM-L-DOPA (181 [98, 333] mM) were similar to those observed in RBE 4 and RBE 4B cells. However, because brain endothelial capillary cells are endowed with a high COMT activity, it was hypothesised that locally formed 3-OM-L-DOPA might produce a more pronounced inhibition for outward transfer of L-DOPA at the level of the abluminal cell side than that occurring at the luminal cell border by circulating 3-OM-L-DOPA. In order to test this possibility some experiments were performed in cells cultured on polycarbonate filters in the presence of S-adenosyl-L-methionine, the methyl donor, L-DOPA being applied from the luminal (apical) cell side. The abluminal fractional outflow is a measure of the extru-
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sion capacity for intracellular substrates at the level of the abluminal cell side. It was quite surprising to observe such a high capacity for extruding intracellular substrates at the level of the abluminal membrane (around 50% of the amount which was taken up). Tolcapone, the COMT inhibitor, abolished the formation of 3-OM-L-DOPA, and the abluminal fractional outflow of L-DOPA in the absence of tolcapone was similar to that observed in the presence of the inhibitor. Furthermore, basal fractional outflow of LDOPA was similar to that for 3-OM-L-DOPA, despite the marked difference in the intracellular concentration between the two substrates. This suggests that 3-OM-L-DOPA and L-DOPA were not competing with each other for extrusion at the abluminal cell side. In order to make sure that the high intracellular concentrations of L-DOPA were not affecting the outflow of 3-OM-L-DOPA it was decided to look at the abluminal fractional outflow of 3-OM-L-DOPA in the absence of L-DOPA. Two concentrations of 3OM-L-DOPA were tested (0.5 and 25 mM), and the results show that abluminal fractional outflow of 3OM-L-DOPA did not depend on the concentration of substrate used and was similar to that observed when both L-DOPA and 3-OM-L-DOPA were present. This was particularly evident in the case of RBE 4B cells, though in RBE 4 cells abluminal fractional outflow of 3-OM-L-DOPA was slightly higher in the absence of L-DOPA. Altogether, these results suggest that competition between intracellular L-DOPA and 3-OM-LDOPA for extrusion at the abluminal cell side may be devoid of importance, in comparison with that occurring at the luminal cell side. On the other hand, the abluminal outflow of both L-DOPA and 3-OM-LDOPA were found to be processes of considerable magnitude, which deserve further study for their kinetic characterisation. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and it is suggested that this immortalised cell line of rat capillary cerebral endothelium might constitute an interesting in vitro model for the study of BBB mechanisms, namely those concerning solute and nutrient transfer across the brain capillary endothelium. In addition, it is suggested that once inside the cell L-DOPA and 3-OM-L-DOPA do not interact for extrusion at the abluminal cell side.
Acknowledgements The present work was supported by grant SAU/123/ 96 from Foundation for Science and Technology.
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References Alexander, G.M., Schwartzman, R.J., Grothusen, J.R., Gordon, S.W., 1994. Effect of plasma levels of large neutral amino acids and degree of parkinsonism on the blood-to-brain transport of levodopa in naive and MPTP parkinsonian monkeys. Neurology 44, 1491 – 1499. Audus, K.L., Borchardt, R.T., 1986. Characteristics of the large neutral amino acid transport system of bovine brain microvessel endothelial cell monolayers. J. Neurochem. 47, 484–488. Begley, D.J., Lechardeur, D., Chen, Z.D., et al., 1996. Functional expression of P-glycoprotein in an immortalised cell line of rat brain endothelial cells, RBE4. J. Neurochem. 67, 988–995. Bradford, M.M., 1976. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Analyt. Biochem. 72, 248–254. Cheng, Y.-C., Prusoff, W.H., 1973. Relationship between the inhibition constant (Ki) and the concentration of the inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108. El Hafny, B., Chappey, O., Piciotti, M., Debray, M., Boval, B., Roux, F., 1997. Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line. Neurosci Lett. 236, 107–111. Frankel, J.P., Kempster, P.A., Bovingdon, M., Webster, R., Lees, A.J., Stern, G.M., 1989. The effects of oral protein on the absorption of intraduodenal levodopa and motor performance. J. Neurol. Neurosurg. Psychiatry 52, 1063–1067. Furgeson, M.D., Dill, R.E., Doris, R.L., 1976. Importance of Omethylation in dopamine-induced motor and behavioral phenomena. Brain Res. 105, 163–167. Gervas, J.J., Muradas, V., Bazan, E., Aguado, E.G., de Yebenes, J.G., 1983. Effects of 3-OM-dopa on monoamine metabolism in rat brain. Neurology 33, 278–282. Guttman, M., Leger, G., Cedarbaum, J.M., et al., 1992. 3-Omethyldopa administration does not alter fluorodopa transport into the brain. Ann. Neurol. 31, 638–643. Himori, N., Mishima, K., 1994. The COMT inhibitor tolcapone potentiates the anticataleptic effect of Madopar in MPP( + )-lesioned mice. Experientia 50, 939–942. Linden, I.B., Nissinen, E., Etemadzadeh, E., Kaakkola, S., Mannisto, P., Pohto, P., 1988. Favorable effect of catechol-O-methyltransferase inhibition by OR-462 in experimental models of Parkinson’s disease. J. Pharmacol. Exp. Ther. 247, 289–293. Ma¨nnisto¨, P.T., Kaakkola, S., 1989. New selective COMT inhibitors: useful adjuncts for Parkinson’s disease ? Trends in Pharmacol. Sci. 10, 54–56. Motulsky, H.J., Spannard, P., Neubig, R., 1994. GraphPad Prism (version 1.0). GraphPad Prism Software Inc, San Diego, USA. Nutt, J.G., Fellman, J.H., 1984. Pharmacokinetics of levodopa. Clin. Neuropharmacol. 7, 35–49. Nutt, J.G., Woodward, W.R., Carter, J.H., Trotman, T.L., 1989. Influence of fluctuations of plasma large neutral amino acids with normal diets on the clinical response to levodopa. J. Neurol. Neurosurg. Psychiatry 52, 481–487.
Nutt, J.G., Woodward, W.R., Gancher, S.T., Merrick, D., 1987. 3-O-methyldopa and the response to levodopa in Parkinson’s disease. Ann. Neurol. 21, 584 – 588. Pardridge, W.M., 1995. Transport of small molecules through the blood-brain barrier: biology and methodology. Advanced Drug Delivery Reviews 15, 5 – 36. Pardridge, W.M., Oldendorf, W.H., 1977. Transport of metabolic substrates through the blood-brain barrier. J. Neurochem. 28, 5 – 12. Reches, A., Fahn, S., 1982. 3-O-methyldopa blocks dopa metabolism in rat corpus striatum. Ann. Neurol. 12, 267–271. Reches, A., Mielke, L.R., Fahn, S., 1982. 3-O-methyldopa inhibits rotations induced by levodopa in rats after unilateral destruction of the nigrostriatal pathway. Neurology 32, 887 – 888. Reilly, D.K., Rivera-Calimlim, L., van Dyke, R., 1980. Catechol-Omethyltransferase activity: a determinant of levodopa response. Clin. Exp. Pharmacol. 28, 278 – 286. Rivera-Calimlim, L., Deepak, T., Anderson, R., Joynt, R., 1977. The clinical picture and plasma levodopa metabolite profile of parkinsonian non-responders. Arch. Neurol. 34, 228 – 232. Roux, F., Durieu-Trautmann, O., Chaverot, N., et al., 1994. Regulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells. J. Cell Physiol. 159, 101 – 113. Sanchez del Pino, M.M., Hawkins, R.A., Peterson, D.R., 1992. Neutral amino acid transport by the blood-brain barrier. Membrane vesicle studies. J. Biol. Chem. 267, 25951 – 25957. Sharpless, N.S., Tyce, G.M., Owen, C.A., 1973. Effect of chronic administration of L-DOPA on catechol-O-methyltransferase in rat tissues. Life Sci. 12, 97 – 106. ´ , P.C., 1994. Cell Soares-da-Silva, P., Fernandes, M.H., Pinto-do-O inward transport of L-DOPA and 3-O-methyl-L-DOPA in rat renal tubules. Br. J. Pharmacol. 112, 611 – 615. Soares-da-Silva, P., Serrao, M.P., Vieira-Coelho, M.A., 1998. Apical and basolateral uptake and intracellular fate of dopamine precursor L-dopa in LLC-PK1 cells. Am. J. Physiol. 274, F243– F251. Vieira-Coelho, M.A., Soares-da-Silva, P., 1997. Apical and basal uptake of L-dopa and L-5-HTP and their corresponding amines, dopamine and 5-HT, in OK cells. Am. J. Physiol. 272, F632– F639. Vieira-Coelho, M.A., Soares-da-Silva, P., 1998. Uptake and intracellular fate of L-3,4-dihydroxyphenylalanine, the dopamine precursor in a human intestinal epithelial cells line: Caco-2. Am. J. Physiol. 275, C104 – C112. Wade, L.A., Katzman, R., 1975a. 3-O-methyldopa uptake and inhibition of L-DOPA at the blood-brain barrier. Life Sci. 17, 131– 136. Wade, L.A., Katzman, R., 1975b. Synthetic amino acids and the nature of L-DOPA transport at the blood – brain barrier. J. Neurochem. 25, 837 – 842. Woodward, W.R., Olanow, C.W., Beckner, R.M., et al., 1993. The effect of L-dopa infusions with and without phenylalanine challenges in parkinsonian patients: plasma and ventricular CSF L-dopa levels and clinical responses. Neurology 43, 1704–1708.
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Interaction between L-DOPA and 3-O-methyl-L-DOPA for transport in immortalised rat capillary cerebral endothelial cells P. Gomes, P. Soares-da-Silva * Institute of Pharmacology & Therapeutics, Faculty of Medicine, 4200 Porto, Portugal Accepted 22 February 1999
Abstract The present study aimed to determine the kinetics of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4 and RBE 4B), to define the type of interaction with 3-O-methyl-LDOPA (3-OM-L-DOPA), sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC), N-(methylamino)-isobutyric acid (MeAIB) and sodium. Non-linear analysis of the saturation curves for L-DOPA and 3-OM-L-DOPA revealed in RBE 4 cells Km values (in mM) of 72 (53, 91) and 40 (25, 57) and in RBE 4B cells Km values (in mM) of 60 (46, 74) and 44 (13, 75), respectively. IC50 values for 3-OM-L-DOPA (RBE 4, 642 [542, 759] mM; RBE 4B, 482 [475, 489] mM) obtained in the presence of a nearly saturating (250 mM) concentration of L-DOPA were greater than the corresponding Ki values (RBE 4, 143 [121, 170] mM; RBE 4B, 93 [92, 95] mM) obtained in the presence of a nearly saturating (250 mM) concentration of 3-OM-L-DOPA; this is compatible with a competitive type of interaction between L-DOPA and 3-OM-L-DOPA. Uptake of both L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells was sensitive to BHC with similar IC50 values. MeAIB (up to 2.5 mM) was found not to interfere with the uptake of both L-DOPA and 3-OM-L-DOPA. Uptake of (250 mM) L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the presence of increasing concentrations of sodium (20 – 140 mM). Homogenates of both cell lines were endowed with considerable COMT activity. Incubation of RBE 4 and RBE 4B cells with L-DOPA (25 mM) in the presence of a methyl donor (S-adenosyl-L-methionine) resulted in the formation of 3-OM-L-DOPA; this was abolished by 1 mM tolcapone. The fractional outflow of intracellular L-DOPA through the luminal and abluminal cell side was not affected by the presence of intracellular 3-OM-L-DOPA. The fractional outflow of exogenous 3-OM-L-DOPA applied from the luminal cell border was similar to that observed for 3-OM-L-DOPA with origin in L-DOPA. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and 3-OM-L-DOPA behaves as a competitive inhibitor for the uptake of L-DOPA. This, however, only occurs for luminal cell inward movement but not for abluminal cell outward movement of the substrates. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Brain capillary endothelial cells; L-DOPA; 3-O-Methyl-L-DOPA; Cellular transport
1. Introduction Replenishment therapy in Parkinson’s disease with (L-DOPA), a direct precursor amino acid of dopamine, has been in the past decades the ultimate strategy to activate the failing dopaminergic system in the parkinsonian brain. In advanced stages of the disease, the usefulness of L-DOPA therapy is limited by a variety of motor complications L-3,4-dihydroxyphenylalanine
* Corresponding author. Tel.: + 351-2-595694; fax: + 351-25502402. E-mail address: [email protected] (P. Soares-da-Silva)
that include: peak-dose dyskinesia; end-of-dose or ‘wearing off’ phenomenon; dystonia; ‘on-off’ phenomenon; and dyskinesia. Some of these motor complications, namely the ‘wearing off’ phenomenon have been suggested to be related to kinetic factors that affect the absolute and relative plasma concentrations of L-DOPA and 3-O-methyl-L-DOPA (3-OM-L-DOPA) (Furgeson et al., 1976; Rivera-Calimlim et al., 1977; Nutt and Fellman, 1984). Although the decarboxylation of L-DOPA is believed to represent a major pathway of its metabolism, methylation into 3-OM-L-DOPA by catechol-O-methyltransferase (COMT) assumes particular relevance in
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conditions of AAAD inhibition (Sharpless et al., 1973; Ma¨nnisto¨ and Kaakkola, 1989). There is, however, some controversy over the extent to which circulating 3-OM-L-DOPA may interfere with the therapeutic response to L-DOPA in parkinsonian patients. On one hand, patients with L-DOPA-induced dyskinesias have been reported to present higher plasma concentrations of 3-OM-L-DOPA (Reilly et al., 1980), systemic 3-OML-DOPA decreased brain uptake of [14C]-L-DOPA (Gervas et al., 1983) or reduced response to L-DOPA (Reches and Fahn, 1982; Reches et al., 1982), and COMT inhibitors potentiate the effects of L-DOPA (Linden et al., 1988; Himori and Mishima, 1994). On the other hand, plasma 3-OM-L-DOPA concentrations in parkinsonian patients treated with L-DOPA on a long-term basis reflect daily L-DOPA dosage and do not vary markedly during the day (Nutt et al., 1987). These observations, plus the fact that 3-OM-L-DOPA makes a small contribution to the total concentration of large neutral amino acids (LNAAs) competing with L-DOPA for transport at the blood-brain barrier (Nutt et al., 1987), support the contention that circulating 3-OM-L-DOPA may not be an important determinant of the clinical response to L-DOPA. In agreement with this view is the observation that infusion of 3-OM-LDOPA to produce plasma concentrations (39 mmol/l) equivalent to those seen in patients on chronic L-DOPA therapy failed to alter the blood-brain transfer rate of 6-[18F]-fluoroDOPA (Guttman et al., 1992). Under physiological conditions LNAAs compete with each other for transport into the brain (Pardridge and Oldendorf, 1977; Pardridge, 1995), and the availability of L-DOPA to brain tissue can be compromised by the presence of high levels of LNAAs in plasma (Alexander et al., 1994). LNNAs and L-DOPA are believed to share the same transporters to cross the BBB (Frankel et al., 1989; Nutt et al., 1989; Woodward et al., 1993; Alexander et al., 1994). LNAAs are transported at the level of the BBB by the L-type amino acid transporter, which is sodium-independent and sensitive to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC). The A-type amino acid transporter has also been shown to transport phenylalanine, but this transporter is mainly involved in the transfer of small nonessential amino acids such as alanine and glycine. Differentiation between A- and L-type is based on sodium-dependence and sensitivity to N-(methylamino)-isobutyric acid (MeAIB) and insensitivity to BHC. The ASC-type of amino acid transporter is a sodium dependent transporter and insensitive to both BHC and MeAIB (Wade and Katzman, 1975a,b; Audus and Borchardt, 1986; Sanchez del Pino et al., 1992). The aim of the present study was to define the kinetics of L-DOPA uptake in an immortalised cell line of rat capillary cerebral endothelial cells (clones RBE 4
and RBE 4B) and the type of interaction produced by 3-OM-L-DOPA. This cell line was obtained by transfection of rat brain microvessel endothelial cells with a plasmid containing the E1A adenovirus gene. These cells display a non-transformed endothelial phenotype expressing the brain microvessel-associated enzymes— g-glutamyl-transpeptidase, alkaline phosphatase and Pglycoprotein (Roux et al., 1994; Begley et al., 1996; El Hafny et al., 1997). We have also attempted to characterise the L-DOPA transporter in these cells, namely its sodium dependence and its sensitivity to 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BHC) and N(methylamino)-isobutyric acid (MeAIB).
2. Methods
2.1. Cell culture RBE 4 and RBE 4B clones were kindly supplied by Dr Franc¸oise Roux (INSERM U. 26, Hoˆpital Fernand Widal, Paris, France) and maintained in a humidified atmosphere of 5% CO2, 95% air at 37°C. RBE 4 (passages 25–29) and RBE 4B cells (passages 2– 6) were grown in Minimum Essential Medium/Ham’s F10 (1:1) (Sigma Chemical Co, MO, USA) supplemented with 300 ng/ml neomycine, 10% foetal bovine serum (Sigma), 1 ng/ml basic fibroblast growth factor, 100 U/ml penicillin G, 0.25 mg/ml amphotericin B, 100 mg/ml streptomycin (Sigma) and 25 mM N-2-hydroxyethylpiperazine-N%-2-ethanosulfonic acid (HEPES; Sigma). The cell medium was changed every 2 days, and the cells reached confluence after 3–4 days of incubation. For subculturing, the cells were dissociated with 0.05% trypsin-EDTA (Sigma), diluted 1:7 and subcultured in petri dishes with a 21-cm2 growth area (Costar, Badhoevedorp, The Netherlands). For uptake studies, the cells were seeded in collagen-treated 24-well plastic culture clusters (internal diameter 16 mm, Costar) at a density of 40 000 cells per well (2.0×104 cells/cm2) or, depending on the experiment, onto collagen treated 0.2 mm polycarbonate filter supports (internal diameter 12 mm Transwell, Costar). For 24 h prior to each experiment, the cell medium was free of foetal bovine serum and basic fibroblast growth factor. Experiments were generally performed 2–3 days after cells reached confluence and 6 days after initial seeding and each cm2 contained about 50 mg of cell protein.
2.2. Transport studies On the day of the experiment, the growth medium was aspirated and the cells were washed with Hanks’ medium at 4°C; thereafter, the cell monolayers were preincubated for 30 min in Hank’s medium at 37°C. The Hanks’ medium had the following composition
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(mM): NaCl 137, KCl 5, MgSO4 0.8, Na2HPO4 0.33, KH2PO4 0.44, CaCl2 0.25, MgCl2 1.0, Tris – HCl 0.15 and sodium butyrate 1.0, pH 7.4. In experiments performed in the presence of different concentrations of sodium, NaCl was replaced by an equimolar concentration of choline chloride. The incubation medium also contained benserazide (50 mM), pargyline (100 mM) and tolcapone (1 mM) in order to inhibit the enzymes aromatic L-amino acid decarboxylase, monoamine oxidase and catechol-O-methyltransferase, respectively. During preincubation and incubation, the cells were continuously shaken and maintained at 37°C. Apical uptake was initiated by the addition of 2 ml Hanks’ medium with a given concentration of the substrate (L-DOPA or 3-OM-L-DOPA). Time course studies were performed in experiments in which cells were incubated with 1 mM substrate for 1, 3, 6 and 12 min. Saturation experiments were performed in cells incubated for 6 min with increasing concentrations of L-DOPA (10 – 1000 mM). In experiments designed to study the effects of 3-OM-LDOPA, BHC, and MeAIB upon the uptake of LDOPA, RBE 4 and RBE 4B cells were preincubated for 30 min in the presence of the compounds to be tested. After preincubation, cells were incubated for 6 min in Hanks’ medium with 250 mM L-DOPA. Uptake was terminated by the rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette followed by a rapid wash with cold Hanks’ medium and the addition of 250 ml of 0.2 mM perchloric acid. The acidified samples were stored at 4°C before injection into the high pressure liquid chromatograph for the assay of L-DOPA or 3-OM-LDOPA. In another series of experiments, cells were cultured in polycarbonate supports, the substrates being applied from the apical side of the monolayer. The incubation medium used in this series of experiments was similar to that described above, in some experiments, the medium contained tolcapone (1 mM) in order to inhibit COMT. The upper and lower chambers contained 400 and 1000 ml, respectively. Cells were pre-incubated for 30 min and, thereafter, incubated in the presence of L-DOPA or 3-OM-L-DOPA. [14C]-Sorbitol (0.4 mM) was used to estimate paracellular fluxes and extracellular trapping of substrates. At the end of the incubation, cells were placed on ice and the medium bathing the apical and basal cell borders was collected, acidified with 2 M perchloric acid and stored at 4°C until it was assayed for L-DOPA and 3-OM-L-DOPA. The cells were washed with ice-cold Hanks’ medium and added with 0.2 mM perchloric acid (200 and 1000 ml in the upper and lower chambers, respectively); the acidified samples were stored at 4°C before injection into the high pressure liquid chromatograph for the assay of L-DOPA and 3-OM-L-DOPA.
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2.3. Assay of L -DOPA and 3 -OM-L -DOPA L-DOPA and 3-OM-L-DOPA were quantified by means of high pressure liquid chromatography with electrochemical detection, as previously reported. The high-pressure liquid chromatograph system consisted of a pump (Gilson model 302; Gilson Medical Electronics, Villiers le Bel, France) connected to a manometric module (Gilson model 802 C) and a stainless-steel 5-mm ODS column (Biophase; Bioanalytical Systems, West Lafayette, IN) of 25 cm length; samples were injected by means of an automatic sample injector (Gilson model 231) connected to a Gilson dilutor (model 401). The mobile phase was a degassed solution of citric acid (0.1 mM), sodium octylsulphate (0.5 mM), sodium acetate (0.1 M), EDTA (0.17 mM), dibutylamine (1 mM) and methanol (8% v/v), adjusted to pH 3.5 with perchloric acid (2 M) and pumped at a rate of 1.0 ml/min. Detection was carried out electrochemically with a glassy carbon electrode, an Ag/AgCl reference electrode and an amperometric detector (Gilson model 141); the detector cell was operated at 0.75 V. The current produced was monitored using the Gilson 712 HPLC software. The lower limits for detection of LDOPA and 3-OM-L-DOPA ranged from 350 to 500 fmol.
2.4. Protein assay The protein content of monolayers of OK cells was determined by the method of Bradford (Bradford, 1976), with human serum albumin as a standard.
2.5. Cell water content Cell water content was simultaneously measured in parallel experiments using [14C]inulin as an extracellular marker and tritiated water as a total water marker. Intracellular water, obtained by subtracting extracellular water from total water, was expressed as ml of cell water per mg protein. Subsequently, the cells were solubilised by 0.1% v/v Triton X-100 (dissolved in 5 mM Tris–HCl, pH 7.4) and radioactivity was measured by liquid scintillation counting.
2.6. Cell 6iability Cells were preincubated for 30 min (in the presence of 3-OM-L-DOPA, BHC or MeAIB) at 37°C and then incubated in the absence or the presence of L-DOPA for a further 6 min. Subsequently the cells were incubated at 37°C for 2 min with trypan blue (0.2% w/v) in phosphate buffer. Incubation was stopped by rinsing the cells twice with Hanks’ medium and the cells were examined using a Leica microscope. Under these conditions, more than 95% of the cells excluded the dye.
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2.7. Data analysis Km and Vmax values for the uptake of L-DOPA and 3-OM-L-DOPA, as determined in saturation experiments, were calculated by non-linear regression analysis, using the GraphPad Prism statistics software package (Motulsky et al., 1994). For the calculation of the IC50, the parameters of the equation for one site inhibition were fitted to the experimental data (Motulsky et al., 1994). Ki values were calculated as defined by Cheng and Prusoff (1973) for competitive inhibition. Fractional outflow was calculated using the expression L-DOPAfluid/(L-DOPAfluid + L-DOPAcell) where L-DOPAfluid indicates the amount of L-DOPA (in nmol/mg protein) which reached the apical or the basal chamber and L-DOPAcell (in nmol/mg protein) indicates the amount of L-DOPA accumulated in the cell monolayer. Fractional outflow of 3-OM-L-DOPA was calculated in the same manner as for L-DOPA, with the exception that it was possible to measure 3-OM-LDOPA present in the medium bathing both the apical and basal cell borders. Arithmetic means are given with S.E.M. or geometric means with 95% confidence values. Statistical analysis was done with a one-way analysis of variance (ANOVA) followed by Newman – Keuls test for multiple comparisons. A P-value less than 0.05 was assumed to denote a significant difference.
2.8. Drugs Drugs used were: 2-aminobicyclo(2,2,1)-heptane-2carboxylic acid (BHC; Sigma Chemical Company, St. Louis, MO, USA), L-b-3,4-dihydroxyphenylalanine (Sigma), 3-O-methyl-L-b-3,4-dihydroxyphenylalanine (Sigma), N-(methylamino)-isobutyric acid (MeAIB; Sigma), pargyline hydrochloride (Sigma) and tolcapone (kindly donated by late Professor Mose´ Da Prada, Hoffman La Roche, Basle, Switzerland). 3. Results To determine the rate constant of uptake, cells were incubated with 1 mM L-DOPA or 1 mM 3-OM-L-DOPA for 1, 3, 6 and 12 min (Fig. 1). The accumulation of L-DOPA and 3-OM-L-DOPA increased linearly with time for several minutes. The intracellular L-DOPA and 3-OM-L-DOPA concentration at initial rate of uptake (6 min) was several times larger that which could be expected by passive equilibration of the substrate. In fact, at 6 min incubation, mean intracellular concentration of L-DOPA and 3-OM-L-DOPA (see Table 1) was 40 – 77 times higher than the substrate concentration in the incubation medium (1 mM). The intracellular water content of cell monolayers was 7.090.7 ml/mg protein (n= 5).
Fig. 1. Time course of L-DOPA (closed squares) and 3-OM-L-DOPA (open squares) accumulation in RBE 4 and RBE 4B cells. Cells were incubated at 37°C with 1 mM of either L-DOPA or 3-OM-L-DOPA. The results reflect levels (in pmol/mg protein) of L-DOPA or 3-OM-LDOPA accumulated. Each point represents the mean of four experiments per group; vertical lines show S.E.M.
Thus, in all subsequent experiments designed to determine the kinetic parameters of L-DOPA and 3-OML-DOPA uptake, the cells were incubated for 6 min with increasing concentrations (10–1000 mM) of the substrate. The accumulation of both L-DOPA and 3OM-L-DOPA was found to be dependent on the concentration used and to be saturable at nearly 250 mM (Fig. 2). It is interesting to observe that the accumulation of L-DOPA in RBE 4B cells was twice that for 3-OM-L-DOPA in this cell line and twice that for L-DOPA and 3-OM-L-DOPA in RBE 4 cells. Km and Table 1 Mean intracellular concentrations (mmol/l) of L-DOPA and 3-OM-LDOPA in cultured RBE 4 and RBE 4B cells at 6 min incubation in the presence of non-saturating concentrations (1 mM) of the substrate
RBE 4 cells RBE 4B cells
L-DOPA (mmol/l)
3-OM-L-DOPA (mmol/l)
39.5 90.9 71.0 91.6
76.3 9 0.4 69.8 90.2
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Fig. 2. Accumulation of L-DOPA (closed squares) and 3-OM-LDOPA (open squares) in RBE 4 and RBE 4B cells. Cells were incubated for 6 min at 37°C and increasing concentrations (10 – 1000 mM) of the substrate were applied from the apical border. The results reflect levels (in pmol/mg protein/6 min) of accumulated L-DOPA or 3-OM-L-DOPA. Each point represents the mean of four experiments per group; vertical lines show S.E.M.
Vmax values for L-DOPA and 3-OM-L-DOPA uptake in both RBE 4 and RBE 4B cells are given in Table 2. Accumulated L-DOPA and 3-OM-L-DOPA were found not to be decarboxylated to their corresponding amines, dopamine and 3-methoxytyramine. Table 2 Km and Vmax values for RBE 4 and RBE 4B cells
RBE 4 cells L-DOPA 3-OM-L-DOPA RBE 4B cells L-DOPA 3-OM-L-DOPA
L-DOPA
and 3-OM-L-DOPA uptake in
Km (mM)
Vmax (nmol/mg protein/6 min)
72 (53, 91) 40 (25, 56)
15.99 0.4 15.7 9 0.5
59 (46, 74) 44 (13, 75)
23.69 0.5 12.79 0.7
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Fig. 3. Inhibition curve of L-DOPA uptake by 3-OM-L-DOPA in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 3 shows inhibition curves for 3-OM-L-DOPA obtained in the presence of a nearly saturating (250 mM) concentration of L-DOPA in RBE 4 and RBE 4B cells. As shown in Fig. 3, 3-OM-L-DOPA was found to produce a concentration dependent inhibition of LDOPA uptake with similar magnitude in both cell lines, although Ki values for 3-OM-L-DOPA in RBE 4B cells were significantly different (PB 0.05) from those in RBE 4 cells (Table 3).
Table 3 IC50 and Ki values for inhibition of L-DOPA uptake by 3-OM-LDOPA, determined in the presence of saturating (250 mM) concentrations of the substrate in cultured RBE 4 and RBE 4B cells Inhibitor
IC50 (mM)
Ki (mM)
RBE 4 cells 3-OM-L-DOPA
642 (542, 759)
143 (121, 170)
RBE 4B cells 3-OM-L-DOPA
482 (475, 489)
93 (92, 95)
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Fig. 4. Inhibition curve of L-DOPA uptake by BHC (open symbols) and MeAIB (closed symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 4 shows the effect of BHC and MeAIB on the uptake of a nearly saturating concentration (250 mM) of L-DOPA. As can be observed, BHC exerted a marked inhibitory effect upon L-DOPA uptake in both RBE 4 and RBE 4B cells, with similar Ki values (Table 4). In contrast to what was observed with BHC, MeAIB, the inhibitor of the A-type system for LNAAs Table 4 IC50 and Ki values for inhibition of L-DOPA and 3-OM-L-DOPA uptake by BHC, determined in the presence of saturating (250 mM) concentrations of the substrate in cultured RBE 4 and RBE 4B cells Substrate
IC50 (mM)
Ki (mM)
1177 (944, 1469) 1168 (846, 1614)
263 (211, 329) 161 (117, 223)
799 (678, 942) 1540 (1269, 1868)
155 (131, 182) 230 (190, 280)
RBE 4B cells L-DOPA
3-OM-L-DOPA RBE 4B cells L-DOPA
3-OM-L-DOPA
Fig. 5. Inhibition curve of 3-OM-L-DOPA uptake by BHC (open symbols) and MeAIB (closed symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
transport, was found not to change the uptake of a nearly saturating concentration of L-DOPA in either RBE 4 and RBE 4B cells (Fig. 4). The effects of BHC and MeAIB on the uptake of a nearly saturating concentration (250 mM) of 3-OM-L-DOPA were similar to those observed for L-DOPA. BHC produced a concentration dependent inhibition of 3-OM-L-DOPA accumulation in both RBE 4 and RBE 4B cells (Fig. 5), with Ki values similar to those observed for L-DOPA (Table 4). Similarly, MeAIB at concentrations up to 2.5 mM was devoid of inhibitory effect on the uptake of 3-OM-L-DOPA. To confirm the view that L-DOPA and 3-OM-LDOPA in these cell lines were taken up by the L-type amino acid transporter, another set of experiments tested the sodium dependence of the uptake of these two substrates. As shown in Fig. 6, uptake of a nearly saturating concentration (250 mM) of L-DOPA and 3-OM-L-DOPA in the absence of sodium in the incubation medium was similar to that observed in the pres-
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ence of increasing concentrations of sodium (20–140 mM). The next series of experiments explored the possibility that endogenous 3-OM-L-DOPA in these cells, i.e. formed from taken up L-DOPA, might compete with L-DOPA for outward transfer, namely at the abluminal side of the cell. For this purpose, both cell lines were first screened for COMT activity. The reaction was conducted using cell homogenates in the presence of a methyl donor and increasing concentrations of adrenaline. As can be observed in Fig. 7, both cell lines were found to O-methylate adrenaline, the amount of metanephrine formed was significantly greater (PB 0.05) in RBE 4B cells than in RBE 4 cells. (Vmax in nmol/mg protein/h: RBE 4, 3.79 0.1; RBE 4B, 4.6 9 0.1); Km values (in mM) for COMT in RBE 4 (4.2 [3.6, 4.7] mM) were similar to those in RBE 4B cells (3.6 [2.8, 4.5] mM). The next step performed consisted in the incubation of RBE 4 and RBE 4B cells cultured in
Fig. 7. O-methylation of increasing concentrations of adrenaline (0.1 to 100 mM) in homogenates of RBE 4 and RBE 4B cells. The results are levels (in nmol/mg protein/h) of metanephrine formed from added adrenaline. Symbols represent means of four experiments per group; vertical lines show S.E.M.
Fig. 6. Effect of increasing medium concentrations of sodium (0, 20, 40, 60, 120 and 140 mM) on the uptake of L-DOPA (closed symbols) and 3-OM-L-DOPA (open symbols) in RBE 4 and RBE 4B cells. Symbols represent means of four experiments per group; vertical lines show S.E.M.
polycarbonate supports and the addition of L-DOPA (25 mM, a non-saturating concentration) from the luminal (apical) cell side, in the absence and the presence of tolcapone (1 mM). The parameters monitored in these experiments were the amount of L-DOPA and 3-OM-LDOPA accumulated in the cell and that appearing in the fluid bathing the luminal (only 3-OM-L-DOPA) and abluminal (L-DOPA and 3-OM-L-DOPA) cell sides. As shown in Table 5, both cell lines were able to convert L-DOPA to 3-OM-L-DOPA, though the extent of LDOPA conversion to 3-OM-L-DOPA was a very limited one (3%). Another interesting point is that outward transfer of 3-OM-L-DOPA at the apical cell side was considerably lower than at the basal cell border. In the presence of tolcapone, levels of 3-OM-LDOPA were below the detection limit of the method (9300 fmol/mg protein) and abluminal fractional outflow of L-DOPA was similar to that observed in the absence of COMT inhibition. However, the most interesting finding is that the abluminal fractional outflow of both L-DOPA and 3-OM-L-DOPA was of the same magnitude, despite the 30-fold difference in intracellular levels between the two compounds. In another series of experiments the cells were loaded from the apical cell border with low (0.5 mM) or high (25 mM) concentrations of 3-OM-L-DOPA, in order to know if abluminal fractional outflow rates might be influenced by the intracellular concentration of the substrate. As shown in Table 6, the intracellular concentration of 3-OM-LDOPA and the amount present in the fluid bathing the basal cell side depended on the concentration of substrate used, but the abluminal fractional outflow did not depend on the intracellular concentration of the substrate. It is interesting to note that while using low concentrations of substrate the intracellular levels 3-
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Table 5 Levels of L-DOPA and 3-OM-L-DOPA (in nmol/mg protein) in cell monolayers, fluid bathing the upper chamber and the lower chamber and abluminal fractional outflow (%) in RBE 4 and RBE 4B cells loaded with L-DOPA (25 mM) from the apical cell border in the absence and the presence of tolcapone (1 mM). Values are means 9 S.E.M. (n =6)a Tolcapone 1 mM
Control L-DOPA
3-OM-L-DOPA
L-DOPA
RBE 4 cells Upper chamber Cell 13.69 0.4 Lower chamber 10.4 90.2 Basal outflow 43.3 91.2
0.16 90.005 0.42 90.006 0.3290.006 43.09 0.6
12.9 9 0.3 8.5 9 0.3 39.7 9 1.1
RBE 4B cells Upper chamber Cell 13.1 90.8 Lower chamber 24.0 9 1.6 Basal outflow 64.5 9 1.5
0.13 9 0.006 0.289 0.029 0.329 0.014 53.694.0
12.8 90.9 21.2 91.0 62.5 92.3
a Levels of 3-OM-L-DOPA in experiments carried out in the presence of tolcapone were below the detection limit ( 9 300 fmol/mg protein).
OM-L-DOPA were similar to those obtained when the cells where loaded with 25 mM L-DOPA (see Table 5) and the abluminal fractional outflow is in the same order of magnitude.
4. Discussion The results presented here show that RBE 4 and RBE 4B cells transport quite efficiently L-DOPA and
Table 6 Levels of 3-OM-L-DOPA (in pmol/mg protein) in cell monolayers and fluid bathing the lower chamber and abluminal fractional outflow (%) in RBE 4 and RBE 4B cells loaded with 3-OM-L-DOPA (0.5 and 25 mM) from the luminal cell sidea 3-OM-L-DOPA 0.5 mM
25 mM
RBE 4 cells Upper chamber Cell Lower chamber Abluminal outflow
146.39 7.5 286.2911.5 66.1 91.6
5953.99 154.6 14746.4 9 900.9 71.09 1.1
RBE 4B cells Upper chamber Cell Lower chamber Abluminal outflow
281.7930.4 406.4 9 59.0 58.7 9 1.4
9268.6 9491.5 16523.49543.0 64.191.3
a
Values are means 9 S.E.M. (n= 6).
3-OM-L-DOPA through the apical (luminal) cell border and several findings demonstrate that this uptake process is a facilitated mechanism. Firstly, steady-state uptake of non-saturating concentrations of L-DOPA and 3-OM-L-DOPA showed a linear dependence on incubation time. Secondly, at an initial rate of uptake (6 min incubation) the cellular transport of L-DOPA and 3-OM-L-DOPA showed a curvilinear dependence on substrate medium concentration, suggesting that the uptake was saturable. Thirdly, 3-OM-L-DOPA and BHC markedly inhibited the uptake of L-DOPA. Evidence for the efficiency of the L-DOPA and 3-OM-LDOPA transport in RBE 4 and RBE 4B cells is the ratio of substrate concentration in cellular water to medium concentration. The intracellular L-DOPA and 3-OM-L-DOPA concentration at the initial rate of uptake (6 min) was several times (40–77) that which would be expected by passive equilibration of L-DOPA. The sensitivity of L-DOPA and 3-OM-L-DOPA uptake to BHC, but not to MeAIB, supports the view that L-DOPA and 3-OM-L-DOPA inward transfer in RBE 4 and RBE 4B cells is promoted neither by the A- nor the ASC-type of amino acid transporter, but most probably by the L-type amino acid transporter. Further evidence that fits this suggestion is that L-DOPA and 3-OM-LDOPA uptake in RBE 4 and RBE 4B cells is not dependent on sodium. The L-type, for leucine preferring, amino acid transporter is facilitative, sodium independent, and blocked by BHC (Wade and Katzman, 1975a,b; Audus and Borchardt, 1986; Sanchez del Pino et al., 1992; Pardridge, 1995). This suggestion also agrees with data obtained under in vivo experimental conditions where it is shown that L-DOPA transport across the rat BBB is markedly inhibited by BHC, but not by MeAIB (Wade and Katzman, 1975a,b). On the other hand, this may suggest that these cells lack the A-type amino acid transporter, which is also in agreement with data obtained for the BBB under in vivo experimental conditions (Wade and Katzman, 1975a,b, and references within). This would also agree with the observations of Audus and Borchardt (1986) showing that in bovine brain microvessel endothelial cell monolayers leucine uptake is markedly inhibited by L-DOPA, but not by MeAIB. Another point suggesting that L-DOPA and 3-OM-L-DOPA in RBE 4 and RBE 4B cells are transported through the L-type amino acid transporter concerns the similarity of the Km values for the uptake of both substrates and the Ki values for BHC when acting as an inhibitor for substrate uptake. In this respect, it is interesting to note that the Michaelis–Menten constant for BHC transport across the BBB (Km = 0.16 mM) under in vivo experimental conditions has been reported to be slightly lower than that for L-DOPA (Km = 0.34 mM), but similar to that
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for 3-OM-L-DOPA (Km =0.13 mM) (Wade and Katzman, 1975a,b). Km values for leucine uptake in bovine brain microvessel endothelial cell monolayers (Km = 0.18 mM) have also been shown (Audus and Borchardt, 1986) to be similar to those observed in RBE 4 and RBE 4B cells. On the other hand, similarities of the Km values for L-DOPA uptake in RBE 4 and RBE 4B cells and those obtained in rat renal tubular epithelial cells (135–216 mM), LLC-PK1 cells (123 mM), OK cells (14–129 mM) and Caco-2 cells (60 mM) (Soaresda-Silva et al., 1994; Vieira-Coelho and Soares-daSilva, 1997; Soares-da-Silva et al., 1998; Vieira-Coelho and Soares-da-Silva, 1998), strongly suggest that the L-DOPA transporter in brain capillary endothelial cells may be similar to that in renal and intestinal epithelial cells. The similarity of the kinetics for L-DOPA and 3OM-L-DOPA uptake and their sensitivity to BHC suggests that the inhibitory effect of 3-OM-L-DOPA on L-DOPA uptake is of the competitive type. This is also supported by the fact that IC50 values were markedly greater than Ki values, as suggested by Cheng and Prusoff (1973) for competitive inhibition. 3-OM-L-DOPA was found to produce marked inhibition of L-DOPA uptake, in agreement with previous suggestions that this metabolite of L-DOPA may limit the access of the parent compound to the brain, as was found to occur in rat brain (Wade and Katzman, 1975a,b). According to some authors (Nutt et al., 1987; Guttman et al., 1992), this may not be a major limitation for the access of L-DOPA to the brain, since steady-state plasma levels of 3-OM-L-DOPA (39 mmol/ l) in parkinsonian patients treated with L-DOPA make a small contribution to the total concentration of LNAAs competing with L-DOPA for transport at the blood–brain barrier. It is interesting, however, to observe that this concentration of circulating 3-OM-LDOPA is similar to the half saturating concentration in RBE 4 and RBE 4B cells, but below the Ki value for inhibition of L-DOPA uptake. In isolated rat renal tubules (Soares-da-Silva et al., 1994), Ki values for inhibition of L-DOPA uptake by 3-OM-L-DOPA (181 [98, 333] mM) were similar to those observed in RBE 4 and RBE 4B cells. However, because brain endothelial capillary cells are endowed with a high COMT activity, it was hypothesised that locally formed 3-OM-L-DOPA might produce a more pronounced inhibition for outward transfer of L-DOPA at the level of the abluminal cell side than that occurring at the luminal cell border by circulating 3-OM-L-DOPA. In order to test this possibility some experiments were performed in cells cultured on polycarbonate filters in the presence of S-adenosyl-L-methionine, the methyl donor, L-DOPA being applied from the luminal (apical) cell side. The abluminal fractional outflow is a measure of the extru-
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sion capacity for intracellular substrates at the level of the abluminal cell side. It was quite surprising to observe such a high capacity for extruding intracellular substrates at the level of the abluminal membrane (around 50% of the amount which was taken up). Tolcapone, the COMT inhibitor, abolished the formation of 3-OM-L-DOPA, and the abluminal fractional outflow of L-DOPA in the absence of tolcapone was similar to that observed in the presence of the inhibitor. Furthermore, basal fractional outflow of LDOPA was similar to that for 3-OM-L-DOPA, despite the marked difference in the intracellular concentration between the two substrates. This suggests that 3-OM-L-DOPA and L-DOPA were not competing with each other for extrusion at the abluminal cell side. In order to make sure that the high intracellular concentrations of L-DOPA were not affecting the outflow of 3-OM-L-DOPA it was decided to look at the abluminal fractional outflow of 3-OM-L-DOPA in the absence of L-DOPA. Two concentrations of 3OM-L-DOPA were tested (0.5 and 25 mM), and the results show that abluminal fractional outflow of 3OM-L-DOPA did not depend on the concentration of substrate used and was similar to that observed when both L-DOPA and 3-OM-L-DOPA were present. This was particularly evident in the case of RBE 4B cells, though in RBE 4 cells abluminal fractional outflow of 3-OM-L-DOPA was slightly higher in the absence of L-DOPA. Altogether, these results suggest that competition between intracellular L-DOPA and 3-OM-LDOPA for extrusion at the abluminal cell side may be devoid of importance, in comparison with that occurring at the luminal cell side. On the other hand, the abluminal outflow of both L-DOPA and 3-OM-LDOPA were found to be processes of considerable magnitude, which deserve further study for their kinetic characterisation. It is concluded that RBE 4 and RBE 4B cells are endowed with the L-type amino acid transporter through which L-DOPA and 3-OM-L-DOPA can be taken up, and it is suggested that this immortalised cell line of rat capillary cerebral endothelium might constitute an interesting in vitro model for the study of BBB mechanisms, namely those concerning solute and nutrient transfer across the brain capillary endothelium. In addition, it is suggested that once inside the cell L-DOPA and 3-OM-L-DOPA do not interact for extrusion at the abluminal cell side.
Acknowledgements The present work was supported by grant SAU/123/ 96 from Foundation for Science and Technology.
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