Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells

B. Sampaio-Maia, M. P. Serrão, and P. Soares-da-Silva

Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the nature and regulation of the inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of L-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases in L-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 µM) and A-23187 (1 and 3 µM) failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells. We concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.

L-3,4-dihydroxyphenylalanine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DOPAMINE IS A WELL-KNOWN NEUROTRANSMITTER and is considered to play an important role in the central nervous system. The concentration of this amine was demonstrated to be dependent on its precursor amino acid level in mammalian brain (13). L-3,4-Dihydroxyphenylalanine (L-DOPA), the immediate precursor of dopamine, has been in the past decades the ultimate strategy to activate the dopaminergic systems in the parkinsonian brain. The supply of L-DOPA to the brain as a means to increase levels of dopamine in brain is justified by the fact that the amine does not cross the blood-brain barrier (BBB).

In several epithelia and at the level of brain capillary endothelium, L-DOPA and other large neutral amino acids are transported by the L-type amino acid transporter. This is a sodium-independent and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BHC)-sensitive transporter. Differentiation between A- and L-type transporters is based on sodium dependence and sensitivity to N-(methylamino)isobutyric acid (MeAIB) and insensitivity to BHC (2, 25, 32, 40). After it has crossed the BBB, L-DOPA is taken into dopaminergic neurones, where it is subsequently converted to dopamine by aromatic L-amino acid decarboxylase. However, the available information regarding the transport systems involved in L-DOPA uptake in dopaminergic neurones is almost nonexistent. The reason for this lack of knowledge derives mainly from technical limitations for the study of L-DOPA transport in neuronal cells. Most studies on the uptake of L-DOPA have been performed with the use of animal models under in vivo experimental conditions, which presents limitations for the detailed investigation of the L-DOPA uptake at the cellular level. On the other hand, studies on L-DOPA uptake in cultured mesenphalic and striatal neurones have been performed mainly to evaluate the neurotoxicity of L-DOPA (1, 7, 16, 18, 19, 21, 24, 35) and were not intended to detail its uptake mechanism(s).

L-DOPA contacts several types of cells before being taken up by dopaminergic neurones on its way from blood vessels to brain neurones, first with endothelial cells of brain capillaries and then with glial cells. Astrocytes, which form a vast cellular network between neurones and blood vessels, are probably the most important type of glial cells concerning communication between blood vessels and neurones. The essential characteristics of the astrocytic processes are their termination on the walls of brain capillaries in astrocytic end feet, forming a palisade between neurones and endothelial cells. Though the astrocytic end feet do not form the BBB, they have an important role in its development and maintenance. Again, though L-DOPA almost certainly contacts this type of cell, it is not known which transport system is involved in the handling of L-DOPA.

The present study was aimed to comparatively evaluate the nature of the transporter involved in the uptake of L-DOPA in cells that are believed to have the most determinant role in the handling of L-DOPA in the brain: brain capillary endothelial cells, astrocytes, and neuronal cells. To define the nature of the transporters involved in the uptake of L-DOPA, we examined the effect of maneuvers that affect cellular sodium and proton gradients and the sensitivity to inhibitors of amino acid transport and sodium. Thereafter, to gain an insight into the molecular mechanisms governing L-DOPA uptake, we evaluated the results of maneuvers that interfere with protein kinase A (PKA)-, protein kinase C (PKC)-, protein kinase G (PKG)-, Ca2+/calmodulin-, and protein tyrosine kinase (PTK)-mediated pathways. As a source of brain capillary endothelial cells, we used RBE4 cells, an immortalized cell line of rat capillary cerebral endothelial cells. These cells were obtained by transfection of rat brain microvessel endothelial cells with a plasmid containing the E1A adenovirus gene, and they display a nontransformed endothelial phenotype expressing the brain microvessel-associated enzymes gamma -glutamyl transpeptidase (gamma -GTP), alkaline phosphatase (ALP), P-glycoprotein and inducible nitric oxide synthase (3, 11, 22, 23, 29), and the GLUT-1 isoform responsible for glucose transport (28, 29). gamma -GTP and ALP are membrane-bound enzymes probably involved in the transport process and are specifically localized in brain endothelial cells. Freshly isolated brain microvessels contain a high activity of gamma -GTP and ALP that is rapidly lost in primary cultures of endothelial cells. RBE4 cells remain sensitive to angiogenic stimuli and astroglial factors regarding the expression of the BBB gamma -GTP and ALP enzymatic activities (28, 29). As a source of astrocytes, we used DI TNC1 cells that were established from cultures of primary type 1 astrocytes from brain diencephalon tissue from 1-day-old Sprague-Dawley rats. The cultures were transfected 3 days after they were initially plated with a DNA construct containing the oncogenic early region of SV40 under the transcriptional control of the human GFAP promoter (pGFA-SV-Tt) and pPGK-neo, which contains the murine phosphoglycerate kinase gene promoter. These cells retain characteristics consistent with the phenotype of type 1 astrocytes (27). As a source of neuronal cells, we used Neuro-2a cells, from a cell line with its origin in mouse neuroblastoma cells, which have been used as an in vitro neuronal model (5, 26, 33).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. The RBE4 cell line was kindly supplied by Dr. Françoise Roux (Institut National de la Santé et de la Recherche Médicale Unité 26, Hôpital Fernand Widal, Paris, France) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. RBE4 cells (passages 27-38) were grown in minimum essential medium-Ham's F-10 (1:1; Sigma, St. Louis, MO) supplemented with 300 ng/ml neomycine, 10% fetal bovine serum (Sigma), 1 ng/ml basic fibroblast growth factor, 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), and 25 mM HEPES (Sigma). The cell medium was changed every 2 days, and the cells reached confluence 3-4 days after the initial seeding. For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:5, and subcultured in Costar petri dishes with 21 cm2 of growth area (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/well (2.0 × 104 cells/cm2). For 24 h before each experiment, the cell medium was free of fetal bovine serum and basic fibroblast growth factor. Experiments were generally performed 2-3 days after cells reached confluency and 5-7 days after the initial seeding, and each square centimeter contained ~50 µg of cell protein.

DI TNC1 and Neuro-2a cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. DI TNC1 cells (ATCC CRL-2005; passages 5-9) were grown in Dulbecco's modified Eagle's medium (Sigma) adjusted to contain 1.5 g/l sodium bicarbonate (Sigma). Neuro-2a cells (ATCC CCL-131; passages 170-181) were grown in minimum essential medium adjusted to contain 1.0 mM sodium pyruvate (Sigma). Both mediums were supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), and 25 mM HEPES (Sigma). The cell medium was changed every 2 days, and the cells reached confluence 3-4 days after the initial seeding. For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:5, and subcultured in Costar petri dishes with 21 cm2 of growth area. 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/well (2.0 × 104 cells/cm2). For 24 before each experiment, the cell medium was free of fetal bovine serum. Experiments were generally performed 2-3 days after cells reached confluency and 5-7 days after the initial seeding, and each square centimeter contained ~50 µg of cell protein.

Transport studies. On the day of the experiment, the growth medium was aspirated and the cells were washed with Hanks' medium; thereafter, the cell monolayers were preincubated for 30 min in Hanks' medium at 37°C. The Hanks' medium had the following composition (in mM): 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1.0 MgCl2, 0.15 Tris · HCl, and 1.0 sodium butyrate, pH 7.4. The incubation medium also contained benserazide (1 µM) and tolcapone (1 µM) to inhibit the enzymes aromatic L-amino acid decarboxylase and catechol-O-methyltransferase, respectively. Time-course studies were performed in experiments in which cells were incubated with 500 µM substrate for 1, 3, 6, 12, and 30 min. Saturation experiments were performed in cells incubated for 6 min with increasing concentrations of L-DOPA (2.5-500 µM). Test substances were applied from the apical side and were present during the preincubation and incubation periods. While the effects of sodium and pH on L-DOPA uptake were studied, preincubation was performed with the Hanks' medium containing 137 mM NaCl at pH 7.4. In experiments in which sodium in the Hanks' medium was reduced from 140 to 120, 60, and 0 mM, osmolarity was maintained by the addition of equimolar concentrations of choline chloride. During preincubation and incubation, the cells were continuously shaken and maintained at 37°C. Apical uptake was initiated by the addition of 2 ml of Hanks' medium with a given concentration of the substrate (2.5 µM L-DOPA) for 6 min. 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 µl of 0.2 mM perchloric acid. The acidified samples were stored at 4°C before being injected into the high-pressure liquid chromatograph for the assay of L-DOPA.

Assay of L-DOPA. L-DOPA was quantified by means of high-pressure liquid chromatography with electrochemical detection, as previously reported (34). The high-pressure liquid chromatograph system consisted of a pump (model 302; Gilson Medical Electronics, Villiers le Bel, France) connected to a manometric module (model 802 C; Gilson) and a stainless steel 5-µm ODS column (Biophase; Bioanalytical Systems, West Lafayette, IN) of 25 cm in length; samples were injected by means of an automatic sample injector (model 231; Gilson) connected to a dilutor (model 401; Gilson). The mobile phase was a degassed solution of citric acid (0.1 mM), sodium octylsulfate (0.5 mM), sodium acetate (0.1 M), EDTA (0.17 mM), dibutylamine (1 mM), and methanol (8% vol/vol), adjusted to pH 3.5 with perchloric acid (2 M) and pumped at a rate of 1.0 ml/min. The detection was carried out electrochemically with a glassy carbon electrode, an Ag-AgCl reference electrode, and an amperometric detector (model 141; Gilson); the detector cell was operated at 0.75 V. The current produced was monitored with the use of Gilson 712 HPLC software. The quantification of L-DOPA was performed by injecting 50-µl aliquots of filtered samples directly into the chromatograph. The lower limits for detection of L-DOPA ranged from 350 to 500 fmol, and responses to the external standard were linear from 0.5 to 50,000 pmol.

Assay of cAMP. cAMP was determined with an enzyme immunoassay kit (Assay Designs, Ann Arbor, MI), as previously described (6). Cells were preincubated for 15 min at 37°C in Hanks' medium (see Transport studies) containing 100 µM 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, in the presence or absence of antagonists. Cells were then incubated for 15 min with forskolin (10 µM) alone or in the presence of thapsigargin (1 µM) or A-23187 (3 µM). At the end of the experiment, the reaction was stopped by the addition of 0.1 M HCl. Aliquots were taken for the measurement of total cAMP content.

Protein assay. The protein content of cell monolayers was determined by the method of Bradford (4), with human serum albumin as a standard.

Cell viability. Cells were preincubated for 30 min at 37°C and then incubated in the absence or the presence of L-DOPA and test compounds for a further 6 min to evaluate potential cell-damaging effects of test substances. Subsequently, the cells were incubated at 37°C for 2 min with trypan blue (0.2% wt/vol) in phosphate buffer. Incubation was stopped by rinsing the cells twice with Hanks' medium, and the cells were examined with a Leica microscope. Under these conditions, >95% of the cells excluded the dye.

Data analysis. Michaelis-Menten constant (Km) and maximum velocity (Vmax) values for the uptake of L-DOPA, as determined in saturation experiments, were calculated from nonlinear regression analysis by using the GraphPad Prism statistics software package (20). Data are given as arithmetic means with SE or geometric means with 95% confidence intervals. Statistical analysis was performed by one-way analysis of variance, followed by the Newman-Keuls test for multiple comparisons. A P value <0.05 was assumed to denote a significant difference.

Drugs. BHC, benserazide, calmidazolium, chelerythrine chloride, cholera toxin, cAMP sodium salt, cGMP sodium salt, forskolin, genistein, genistin, IBMX, L-DOPA, phorbol 12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), 4alpha -phorbol 12,13-didecanoate (PDDC), sodium nitroprusside, trifluoperazine dihydrochloride, trypan blue, tyrphostin 1, and tyrphostin 25 were purchased from Sigma. LY-83583 [6-(phenylamino)-5,8-quinolinedione] and zaprinast were obtained from Research Biochemicals International (Natick, MA) and May & Backer (Dorset, UK), respectively. Tolcapone was kindly donated by late Prof. Mosé Da Prada (Hoffman La Roche, Basel, Switzerland).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To determine initial rates of uptake, we incubated cells with a saturating (500 µM) concentration of L-DOPA for 3, 6, 12, and 30 min. In all three types of cells (RBE4, DI TNC1, and Neuro-2a), uptake of a saturating concentration of L-DOPA (500 µM) was linear with time for up to 30 min of incubation (Fig. 1). In a subsequent set of experiments designed to determine the kinetics of the L-DOPA transporter, cells were incubated at 37 and 4° C for 6 min with increasing concentrations (2.5-500 µM) of the substrate (Fig. 2). In all three types of cells, the accumulation of L-DOPA was found to be dependent on the concentration and to be saturable at 250 µM (Fig. 2). As shown in Fig. 2, uptake of L-DOPA was largely dependent on the temperature, a finding particularly evident in RBE4 and Neuro-2a cells. The specific uptake of L-DOPA in Neuro-2a cells was higher than in RBE4 and DI TNC1 cells (Fig. 2 and Table 1). Kinetic parameters of L-DOPA uptake (Km and Vmax) were determined by nonlinear analysis of the specific analysis of saturation curve for L-DOPA and are given in Table 1. As shown in Table 1, the affinity of the transporter for L-DOPA did not differ among the three types of cells, as evidenced by similar Km values.


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Fig. 1.   Time course of L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation in RBE4, DI TNC1, and Neuro-2a cells. Cells were incubated at 37°C with 500 µM L-DOPA, and results reflect levels of substrate accumulated. Data represent means ± SE of 4 experiments per group.



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Fig. 2.   Concentration-dependent accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells. Cells were incubated for 6 min at 37 and 4°C, and increasing concentrations (2.5-500 µM) of the substrate were applied from the apical cell side. Results reflect levels of accumulated L-DOPA. Data represent means ± SE of 4-8 experiments per group.


                              
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Table 1.   Km and Vmax values for L-DOPA uptake in RBE 4, DI TNC1, and Neuro-2A cells under control conditions and in the presence of BHC or calmidazolium

In experiments designed to define the nature of the transporter and its molecular regulation, we studied uptake at nonsaturating concentrations of L-DOPA (2.5 µM), which at 4°C was less than 10% of that occurring at 37°C (Table 2). To examine the nature of the transporter, we decided to evaluate first the sodium requirements for L-DOPA uptake and then its sensitivity to inhibitors of amino acid transporters. Reducing extracellular sodium (from 140 to 120, 60, and 0 mM) did not affect the accumulation of L-DOPA in DI TNC1 cells (Table 2). However, in RBE4 and Neuro-2a cells, reducing extracellular sodium produced slight, but significant (P < 0.05), reductions in the accumulation of L-DOPA, a finding particularly evident in RBE4 cells. Maneuvers that affect transepithelial flux of sodium, such as the addition of amphotericin B (2.5 µg/ml), amiloride (100 µM), or ouabain (500 µM), failed to largely affect L-DOPA accumulation in Neuro-2a cells, whereas in DI TNC1 cells, ouabain produced a significant decrease (28% reduction) in L-DOPA accumulation (Table 2). In RBE4 cells, amphotericin B, amiloride, and ouabain produced slight, but statistically significant, decreases (5-15% reductions) in L-DOPA accumulation. MeAIB (1 mM) failed to affect the uptake of L-DOPA, whereas BHC produced concentration-dependent inhibition of L-DOPA uptake in all three cell types [geometric means (95% confidence intervals): RBE4, IC50 = 140 (107, 184) µM; DI TNC1, IC50 = 405 (151, 1,091) µM; Neuro-2a, IC50 = 117 (93, 148) µM] (Fig. 3). In all three types of cells, the inhibitory effect of BHC (RBE4, 100 µM; DI TNC1, 300 µM; Neuro 2 A, 80 µM) on the accumulation of L-DOPA was of the competitive type, as evidenced by increases in Km and decreases in Vmax values for L-DOPA uptake (Table 1 and Fig. 4). Together, these results suggest that the inward transfer of L-DOPA in all three types of cells may be largely promoted through the BHC-sensitive and sodium-independent L-type amino acid transporter, despite differences in their sensitivity to BHC. The activity of the mammalian system L in various cell types and tissues has been reported to be dependent on extracellular pH (9). As shown in Fig. 5, the accumulation of L-DOPA in all three types of cells was significantly (P < 0.05) higher at an acidic pH.

                              
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Table 2.   Effect of maneuvers that affect energy-dependent processes and transepithelial flux of sodium ions and modulators of PKA, PKG, PKC, and PTK on the uptake of L-DOPA



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Fig. 3.   Effect of N-(methylamino)isobutyric acid (MeAIB) and 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BHC) on the accumulation of L-DOPA (2.5 µM) in RBE4, DI TNC1, and Neuro-2a cells. Values are percent control for accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells (absolute levels: RBE4, 831 ± 12 pmol/mg protein, n = 10; DI TNC1, 510 ± 49 pmol/mg protein, n = 7; Neuro-2a, 1,623 ± 132 pmol/mg protein, n = 10). [Inhibitor], inhibitor concentration. Data represent means ± SE of 4-10 experiments per group.



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Fig. 4.   Effect of BHC and calmidazolium on the concentration-dependent accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells. Cells were incubated for 6 min at 37°C, and increasing concentrations (2.5-500 µM) of the substrate were applied from the apical cell side. Results reflect levels of accumulated L-DOPA. Data represent means ± SE of 4-8 experiments per group.



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Fig. 5.   Effect of pH on the accumulation of L-DOPA (2.5 µM) in RBE4, DI TNC1, and Neuro-2a cells. Values are percent control for accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells (absolute levels: RBE4, 657 ± 61 pmol/mg protein, n = 8; DI TNC1, 600 ± 107 pmol/mg protein, n = 8; Neuro-2a, 1,415 ± 72 pmol/mg protein, n = 8). Data represent means ± SE of 8 experiments per group.

The next series of experiments explored the role of intracellular regulatory pathways in the cellular handling of L-DOPA. Involvement of a PKA-mediated pathway in the regulation of L-DOPA uptake was tested by examining the effect of pretreating cells for 30 min with compounds that are known to increase intracellular cAMP levels. Dibutyryl cAMP (1 mM), the adenylyl cyclase stimulant forskolin (100 µM), the phosphodiesterase inhibitor IBMX (1 mM), and cholera toxin (3 µg/ml) failed to affect the accumulation of a nonsaturating (2.5 µM) concentration of L-DOPA in RBE4 and Neuro-2a cells (Table 2). By contrast, DI TNC1 cells were particularly sensitive to the stimulatory effects of forskolin and IBMX produced on L-DOPA accumulation and also, to a minor but significant extent, to dibutyryl cAMP (Table 2).

In another series of experiments, we tested the involvement of a PKG-mediated pathway in the regulation of L-DOPA uptake. In RBE4 and Neuro-2a cells, cGMP (1 mM), the selective cGMP phosphodiesterase inhibitor zaprinast (30 µM), the guanylyl cyclase inhibitor LY-83583 (30 µM), and sodium nitroprusside (100 µM) failed to affect the accumulation of a nonsaturating concentration L-DOPA (Table 2). On the other hand, in DI TNC1 cells LY-83583 produced a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation (Table 2).

The possible role of PKC in the regulation of L-DOPA uptake in RBE4, DI TNC1, and Neuro-2a cells was tested by examining the effect of pretreating cells with either PKC activators or inhibitors. The PKC activators PDBu (1 µM) and PMA (5 µM), the inactive phorbol ester PDDC (1 µM), and the PKC inhibitor chelerythrine (10 µM) failed to affect the accumulation of L-DOPA (Table 2). RBE4 and DI TNC1 cells constituted one exception, in which chelerythrine was found to produce a significant increase in L-DOPA accumulation (Table 2).

In another study, we tested the involvement of PTK in the regulation of L-DOPA uptake by RBE4, DI TNC1, and Neuro-2a cells. Basically, the effects of PTK inhibitors genistein and tyrphostin 25 were found not to differ from those exerted by their negative controls genistin and tyrphostin 1, suggesting the lack of involvement of PTK on the regulation of L-DOPA accumulation (Table 2).

In the final series of experiments, the role of Ca2+/calmodulin-mediated pathways in the regulation of L-DOPA by RBE4, DI TNC1, and Neuro-2a cells was tested by examining the effect of pretreating the cells with the calmodulin inhibitors calmidazolium and trifluoperazine. Both compounds produced concentration-dependent inhibition of L-DOPA (2.5 µM) uptake (Fig. 6), with IC50 values in the same range of magnitude (Table 3). The inhibitory effect of calmidazolium on the accumulation of L-DOPA by RBE4, DI TNC1, and Neuro-2a cells was of the noncompetitive type, as evidenced by the decrease in Vmax without changes in Km values for L-DOPA uptake (Table 1 and Fig. 4). The calcium ionophore A-23187 (calcimicin) and thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump (17, 37), failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells (Fig. 7).


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Fig. 6.   Effect of calmidazolium (A) and trifluoperazine (B) on accumulation of L-DOPA (2.5 µM) in RBE4, DI TNC1, and Neuro-2a cells. Values are percent control for accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells (absolute levels: RBE4, 792 ± 43 pmol/mg protein, n = 11; DI TNC1, 652 ± 29 pmol/mg protein, n = 13; Neuro-2a, 1,253 ± 39 pmol/mg protein, n = 10). Data represent means ± SE of 4-8 experiments per group.


                              
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Table 3.   IC50 values for inhibition of L-DOPA uptake by calmidazolium and trifluoperazine, determined in the presence of nonsaturating (2.5 µM) concentrations of L-DOPA in cultured REB 4, DI TNC1, and Neuro-2A cells



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Fig. 7.   Effect of A-23187 (A) and thapsigargin (B) on accumulation of L-DOPA (2.5 µM) in RBE4, DI TNC1, and Neuro-2a cells. Values are percent control for accumulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells (absolute levels: RBE4, 964 ± 21 pmol/mg protein, n = 6; DI TNC1, 413 ± 9 pmol/mg protein, n = 8; Neuro-2a, 1,183 ± 57 pmol/mg protein, n = 6). Data represent means ± SE of 4-8 experiments per group.

Because certain adenylyl cyclases in some cells are activated by Ca2+/calmodulin, while others are inhibited by Ca2+ (36), we decided to evaluate in more detail the relationship between Ca2+ and cAMP in the cellular handling of L-DOPA. For this purpose, we felt it worthwhile to evaluate the effect of A-23187 and thapsigargin on cAMP accumulation under steady-state conditions and when challenged with forskolin. As shown in Fig. 8, forskolin (10 µM) produced a marked increase in cAMP levels in all three types of cells that was significantly reduced by thapsigargin but not by A-23187 (Fig. 8). The basal levels of cAMP levels were not affected by thapsigargin or A-23187 (data not shown).


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Fig. 8.   Effect of A-23187 (3 µM) and thapsigargin (1 µM) on forskolin (10 µM)-stimulated accumulation of cAMP in RBE4, DI TNC1, and Neuro-2a cells. Values are absolute levels of cAMP. Data represent means ± SE of 4 experiments per group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results presented show that RBE4, DI TNC1, and Neuro-2a cells transport L-DOPA quite efficiently, and several findings demonstrate that this uptake process is a facilitated mechanism. First, uptake of saturating concentrations of L-DOPA showed a linear dependence on incubation time. Second, at an initial rate of uptake (6-min incubation), the cellular transport of L-DOPA showed a curvilinear dependence on substrate medium concentration, suggesting that the uptake was saturable. Third, low temperature and Ca2+/calmodulin inhibitors, calmidazolium and trifluoperazine, markedly inhibited the uptake of L-DOPA.

The sensitivity of L-DOPA uptake to BHC, but not to MeAIB, supports the view that L-DOPA inward transfer in RBE4, DI TNC1, and Neuro-2a cells is promoted not by either the A- or ASC-type amino acid transporter but, most probably, by the L-type amino acid transporter. Although most L-DOPA was entering the cells in a sodium-independent manner, a minor component of L-DOPA uptake was found to require extracellular sodium (RBE4 and Neuro-2a cells). Maneuvers that alter sodium gradients, such as those resulting from the application of amiloride (an inhibitor of the Na+/H+ exchanger), ouabain (an inhibitor of the Na+-K+-ATPase), and amphotericin B (a sodium ionophore), were also found not to markedly affect L-DOPA uptake. This was particularly evident in Neuro-2a cells, whereas in RBE4 and DI TNC1 cells amiloride, ouabain, and amphotericin B were found to produce a slight but significant reduction in L-DOPA accumulation. Together, these results agree with the suggestion that L-DOPA was taken up mainly through the L-type amino acid transporter (LAT1 or LAT2) and that a minor amount might enter the cell through the y+ LAT1, a sodium-dependent transporter (38, 39). The L-type (leucine preferring) amino acid transporter is facilitative, sodium independent, and blocked by BHC but not by MeAIB (2, 25, 32). Another point suggesting that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter concerns the similarity of Km values for L-DOPA uptake and inhibition constant (Ki) values for BHC when acting as an inhibitor for L-DOPA uptake. The finding that only Km values, but not Vmax values, for L-DOPA uptake were changed when saturation experiments were performed in the presence of a concentration of the inhibitor equal to IC50 values, suggests that the type of inhibition by BHC on L-DOPA uptake is of the competitive type. Other evidence that fits the view that L-DOPA in these three types of cells is promoted through the L-type amino acid transporter is that accumulation of L-DOPA was significantly higher at an acidic pH. In fact, the activity of the mammalian system L in various cell types and tissues has been reported to be dependent on extracellular pH (9).

After defining the mechanism of uptake, we examined the regulation of L-DOPA in RBE4, DI TNC1, and Neuro-2a cells. We concentrated on intracellular regulatory pathways that have been shown to play an important role in the regulation of uptake of other substrates by epithelial cells (PKA-, PKG-, PKC-, PTK-, and Ca2+/calmodulin-mediated pathways). Using specific modulators of these pathways, we found that PKA-, PKG-, PKC-, and PTK-mediated pathways appear to have no role in regulating L-DOPA uptake in RBE4 and Neuro-2a cells. However, two types of evidence suggest that this may be not the case in DI TNC1 cells. In fact, these cells were found to accumulate more L-DOPA after exposure to both forskolin and IBMX, suggesting that increases in cAMP may enhance the uptake or the accumulation of L-DOPA. The slight increase produced by dibutyryl cAMP may be related to its limited cellular permeability. On the other hand, compounds that are antagonists of Ca2+/calmodulin-mediated pathways, such as calmidazolium and trifluoperazine, caused a significant and concentration-dependent reduction in L-DOPA uptake in all three types of cells. The inhibitory effect of calmidazolium was accompanied by a marked decrease in Vmax values without changes in Km values, which is compatible with a noncompetitive inhibitory profile. This would suggest that calmidazolium might reduce the number of L-type amino acid transporters in the apical membrane. Similar findings were observed in human renal and intestinal epithelial cells, where calmidazolium and other inhibitors of Ca2+/calmodulin-mediated pathways, such as trifluoperazine and KN-62, have been found to markedly reduce riboflavin accumulation (14, 30, 31). An increase in intracellular calcium, on the other hand, appears to play no role in the regulation of L-DOPA uptake in RBE4 and Neuro-2a cells, as evidenced by the lack of effect of A-23187 and thapsigargin. By contrast, DI TNC1 cells responded to A-23187 and thapsigargin with marked increases in L-DOPA accumulation.

From a conceptual point of view, and considering the similarities and differences among these three types of cells, it appears that L-DOPA in RBE4 and Neuro-2a cells is taken up by the L-type amino acid transporter, which is under the control of calmodulin-mediated pathways. On the other hand, astrocytes, exemplified here by DI TNC1 cells, appear to handle L-DOPA in a more complex manner. As in endothelial and neuronal cells, astrocytes take up L-DOPA through the L-type amino acid transporter, which is under the control of calmodulin-mediated pathways. However, other processes appear to regulate the accumulation of L-DOPA in astrocytes. Increases in intracellular Ca2+, as induced by A-23187 and thapsigargin, and increases in cAMP, as induced by forskolin and IBMX, were accompanied by marked increases in L-DOPA accumulation. It is quite likely that, in astrocytes, processes involved in L-DOPA accumulation (uptake and storage) may respond to increases in Ca2+ and/or cAMP as trigger mechanisms. Because certain adenylyl cyclases in some cells are activated by Ca2+/calmodulin while others are inhibited by Ca2+ (36), we felt it worthwhile to determine the relationship between Ca2+ and cAMP in these three types of cells. In fact, the ability of adenylyl cyclases to be regulated by physiological transitions in Ca2+ provides a key point for integration of cytosolic Ca2+ concentration and cAMP signaling. Consistent with the earlier reports (10, 15), we found that thapsigargin, but not the calcium ionophore A-23187, induced a marked decrease in forskolin-stimulated cAMP accumulation. This finding suggests that RBE4, DI TNC1, and Neuro-2a cells express the Ca2+-inhibitable forms of adenylyl cyclase (types V and VI). On the other hand, these data are also in agreement with the view that Ca2+-sensitive adenylyl cyclases require Ca2+ entry for their regulation of, rather than diffuse elevations in, intracellular Ca2+ (8, 12). However, the finding that the effects of thapsigargin on cAMP (a decrease) were in contrast to those on L-DOPA (an increase) suggests that Ca2+-dependent effects on L-DOPA accumulation were most probably not related to changes in cAMP levels. This suggestion is reinforced by the finding that A-23187 failed to alter cAMP levels, whereas it markedly increased L-DOPA accumulation. Alternatively, one might suggest that the relationship among cAMP, intracellular Ca2+, and L-DOPA entry would result from modulation of changes in intracellular Ca2+ as caused by cAMP. Recently, in fact, capacitive Ca2+ entry in cultured rat cerebellar astrocytes was directly enhanced by an increase in intracellular cAMP (41).

Another mechanism that may play a role in the handling of L-DOPA in astrocytes concerns cGMP and nitric oxide. The nitric oxide donor sodium nitroprusside was found to decrease L-DOPA accumulation, whereas the guanylyl cylase inhibitor LY-83583 produced the opposite effect. Together, these results would suggest that cGMP might tonically reduced uptake of L-DOPA. Because RBE4 and Neuro-2a cells failed to respond to these compounds, one might suggest that cGMP does not directly interfere with the activity of the (L-type amino acid) transporter but interacts with other processes that, in astrocytes, regulate the handling of L-DOPA. These aspects are presently under investigation in our laboratory.

We have concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.


    ACKNOWLEDGEMENTS

This work was supported by Foundation for Science and Technology Grant PRAXIS/SAU/123/96.


    FOOTNOTES

Address for reprint requests and other correspondence: P. Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal (E-mail: patricio.soares{at}mail.telepac.pt).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 23 March 2000; accepted in final form 12 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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