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Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal
1Correspondence: Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal. E-mail: psoaresdasilva{at}netcabo.pt
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: LAT2 • siRNA • LLC-PK1 cells • amino acid exchanger
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. This coupling defect is genetic (precedes the onset of hypertension and cosegregates with the hypertensive phenotype), receptor specific (not shared by other humoral agents), and organ and nephron segment selective (occurs in proximal tubules, but not in cortical collecting ducts or the brain striatum). A consequence of the defective dopamine receptor/adenylyl cyclase coupling in the SHR and hypertensive subjects is a decreased ability to promote natriuresis (1
2
3)
. However, dopamine production and excretion in SHR is normal or even increased compared with that in WKY (4
5
6)
. This would suggest that in SHR the increased ability to form dopamine at the kidney level might correspond to an attempt to overcome the deficient dopamine-mediated natriuresis (1)
, as has been reported for aged Fischer 344 rats (7)
.
Recently, our group demonstrated for the first time that overexpression of LAT2 in the SHR kidney is organ specific and precedes the onset of hypertension, accompanied by an enhanced ability to take up L-DOPA (8)
. It was therefore suggested that overexpression of renal LAT2 might constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natriuresis generally observed in this genetic model of hypertension (8)
. This adaptive mechanism might be limited to renal tissues: at the intestinal level, where defective transduction of the D1 receptor signal also occurs (9)
, it is not accompanied by increases in either dopamine tissue levels (9)
or intestinal LAT2 expression (8)
. LAT1 and LAT2 are two isoforms of system L (for leucine preferring) that convey the Na+-independent transport of large branched and aromatic neutral amino acids. LAT1 is expressed in nonepithelial cells such as brain, spleen, thymus, testis, skin, liver, placenta, skeletal muscle, and stomach (10
, 11)
and has a high affinity for amino acid substrates. The second isoform of system L, LAT2, is highly expressed in polarized epithelia (12)
, suggesting an important role in transepithelial amino acid transport, but it has a lower affinity for amino acid substrates than for LAT1 (12
, 13)
. Another difference between LAT1 and LAT2 concerns their sensitivity to extracellular pH for amino acid uptake (11)
. Recent studies from our laboratory have shown that L-DOPA uptake in renal epithelial cells may be promoted through the L-type amino acid transporter (14)
, as has been found in intestinal epithelial cells (15)
, and at the level of brain capillary endothelium (16
17
18
19
20)
.
The present work aimed to evaluate the presence and define the role of LAT2 amino acid transporter in L-DOPA handling by LLC-PK1 cells, an established epithelial cell line derived from porcine-derived renal tubule epithelial cells that retain several properties of proximal tubular epithelial cells in culture (21)
. To understand the physiological role of LAT2-mediated L-DOPA handling, we used 21-nucleotide small interfering RNA (siRNA) duplexes to specifically suppress LAT2 expression in LLC-PK1 cells, a cell line that takes up L-DOPA largely through Na+-independent transporters (14)
. Because the LAT2 gene from pig has not yet been described, LAT2 gene silencing was performed after cloning the LLC-PK1 LAT2 gene. It is reported that gene silencing with a LAT2 siRNA, but not the mismatch LAT2 siRNA, markedly reduced LAT2 cDNA levels, accompanied by a marked reduction in accumulation of [14C]-L-DOPA and a decrease in the efflux of intracellular [14C]-L-DOPA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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80 µg of cell protein.
RT-PCR
Total RNA was isolated from cell monolayers using TRIZOL® reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. The RNA obtained was dissolved in diethylpyrocarbonate-treated water and quantified by spectrophotometry at 260 nm. Five micrograms total RNA were reverse transcribed to cDNA with SuperScript First-strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions. The cDNA was amplified by PCR using two sets of primers: one degenerate set simultaneously specific for human (GenBank accession #AF104032 forward nt 258-275 and reverse nt 1268-1287), rat (GenBank accession #AB015432forward nt 259-275 and reverse nt 1280-1299), mice (GenBank accession #AB023409 forward nt 204-220 and reverse nt 1225-1244), and pig (GenBank accession #BI184800 and #BI181265 forward nt 296-312 and reverse nt 448-467) LAT1 (forward: 5'-GG(C/T) TCG (G/T)GC ATC TTC GT-3' and reverse: 5'-(G/A)CA (G/C)AG CCA GTT GAA GAA GC-3'); and another, specific for pig (GenBank accession #BG894387 forward nt 11-31 and reverse nt 430-447) LAT2 (forward: 5'-AGG CAA CGA AAC AAC ACT GAA-3' and reverse 5'-AAG CAG GTG GGG AAG AGC-3'). PCR was performed with platinum TaqPCRx DNA polymerase (Invitrogen) with 1x enhancer for LAT1 and LAT2. Amplification conditions were as follows: hot start of 2 min at 95°C; 30 cycles of denaturing (95°C for 45 s), annealing (58°C for 45 s), and extension (68°C for 45 s); and a final extension of 7 min at 68°C. The PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide.
5'RACE (rapid amplification of cDNA ends)
The 5' cDNA end of LAT2 was obtained from total RNA using the kit FirstChoice RLM-RACE from Ambion (Austin, TX, USA) according to the manufacturer’s instructions. The nested PCR reactions were performed using High Fidelity Taq polymerase (Invitrogen). PCR conditions were the same for the outer and inner reactions: hot start of 2 min at 94°C; 30 cycles of denaturing (94°C for 30 s), annealing (60°C for 30 s), extension (68°C for 1.5 min), and a final extension of 7 min at 68°C.
Cloning and sequencing
PCR products obtained (from RT-PCR and 5'RACE) were gel purified and cloned into pCR4-TOPO vector using the TOPO TA cloning kit for sequencing (Invitrogen). TOP10 Escherichia coli cells were electroporated with the recombinant plasmids and transformants were analyzed by colony PCR. Sequencing of isolated clones was performed in both directions by GATC Biotech AG (Konstanz, Germany) using T7 and T3 primers. Nucleotide homology searching was performed against nonredundant and dbEST using basic local alignment tool (BLAST) via online connection to the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). Multiple nucleotide or amino acid sequence comparisons were done with CLUSTALW via online connection to the Pasteur Institute.
LAT2 gene silencing
Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described (22)
. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into certain organisms and cell types causes degradation of the homologous mRNA. One target site within the LAT2 gene was chosen from the pig LAT2 mRNA sequence (Table 1
). Nucleotide homology searching was performed against nonredundant and dbEST using BLAST via online connection to the NCBI. The siRNA, which target nucleotides 97-117 of the pig LAT2 mRNA sequence, and the nonspecific siRNA duplexes containing a 6 base pair (bp) mismatch (Table 1)
were prepared by a transcription-based method using the Silencer siRNA construction kit (Ambion) according to the manufacturer’s instructions. The dsRNAs were complexed with lipofectin (3%) and added to the cells for 12 h in serum-free medium. Thereafter, cells were cultured for 48 h in serum-supplemented medium, which was replaced by serum-free medium 24 h before the experiments. The cells were used 72 h after transfection.
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Real-time quantitative RT-PCR
For real-time quantitative RT-PCR analysis, total RNA was extracted from LLC-PK1 cells using the TRIZOL® reagent according to the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA). First-strand cDNA was produced using SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies) with random hexamer primers. One microgram of total RNA was used for each reverse transcription reaction (20 µL). For the real-time quantitative PCR, 1 µL of the 20 µL reverse transcription reaction mixture was used. For the calibration curve, a 112 bp LAT2 cDNA fragment (from the LLC-PK1 LAT2 sequence described above) was amplified using conventional PCR (forward primer 5'-TCG CTG TGA CTT TTG GAG AGA-3'; reverse primer 5'-CGG GAG GAG GTG AAG AGG-3', corresponding to nt 893-914 and nt 987-1005, respectively), using Platinum TaqPCRx DNA Polymerase (Life Technologies). The PCR products were purified (QIAEX II DNA gel Extraction Kit, Qiagen, Chatsworth, CA, USA) and quantified by spectrophotometry at 260 nm. The DNA concentration was calculated and the DNA was diluted accordingly in serial steps. Real-time PCR was carried out using a LightCycler (Roche, Nutley, NJ, USA). 20 µL reactions were set up in microcapillary tubes using the following final concentrations: 0.5 µM each of LAT2 forward and LAT2 reverse primers, 1x SYBR Green master mix (LightCycler FastStart DNA MasterPlus SYBR Green I, Roche), and 1 µL of cDNA. Cycling conditions were as follows: denaturation (95°C for 15 min), amplification and quantification (95°C for 10 s, 60°C for 10 s, and 72°c for 5 s, with a single fluorescence measurement at the end of the 72°C for 5 s segment) repeated 40 times, a melting curve program (65°C for 15 s and 95°C with a heating rate of 0.1°C/s and continuous fluorescence measurement), and a cooling step to 40°C. Data were analyzed using LightCycler analysis software. The PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide.
Immunoblotting
Cell monolayers were washed with PBS, then lysed in RIPA buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL PMSF, 2 µg/mL leupeptin, and 2 µg/mL aprotinin. Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin (BSA) as standard. Cell lysates were boiled in sample buffer (35 mM Tris-HCl, pH 6.8, 4% SDS, 9.3% dithiothreitol, 0.01% bromophenol blue, 30% glycerol) at 95°C for 5 min. Samples containing 40 µg of cell protein were separated by SDS-PAGE with 10% polyacrylamide gel, then electroblotted onto nitrocellulose membranes (Bio-Rad). Blots were blocked overnight with 5% nonfat dry milk in PBS-T (0.05% Tween 20 in 10 mM PBS) at 4°C with constant shaking. Blots were then incubated with rabbit anti-LAT1 polyclonal antibody (1:500; Serotec, Oxford, UK) in 5% nonfat dry milk in TBS-T for 1.5 h at room temperature. Membranes were washed three times with TBS-T, then incubated with peroxidase-labeled goat anti-rabbit IgG (1:3000; Santa Cruz Biotechnology) for 1.5 h at room temperature and developed to detect the specific protein using enhanced chemiluminescence reagents. Chemiluminescence of immunocomplexes was detected using an ECL kit (Amersham Life, Arlington Heights, IL, USA). Protein concentration was determined using a protein assay kit (Bio-Rad Laboratories) with BSA as standard.
Transport of [14C] L-DOPA
On the day of the experiment, the growth medium was aspirated and cell monolayers were preincubated for 30 min in Hanks’ medium at 37°C. The Hanks’ medium had the following composition (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. The incubation medium also contained benserazide (30 µM) and tolcapone (1 µM) in order to inhibit the enzymes aromatic L-amino acid decarboxylase and catechol-O-methyltransferase, respectively. Apical uptake was initiated by the addition of 1 mL Hanks’ medium with a given concentration of the substrate. Time course studies were performed in experiments in which cells were incubated with 0.25 µM [14C] L-DOPA for 1, 3, 6, 12, 30, and 60 min. Saturation experiments were performed in cells incubated for 6 min with 0.25 µM [14C]-L-DOPA in the absence and presence of increasing concentrations of the unlabeled substrate. In experiments performed in the presence of different concentrations of sodium, sodium chloride was replaced by an equimolar concentration of choline chloride. In experiments performed at different pH values, pH of Hanks’ medium was adjusted to the desired pH value with 2 M HCl or 1 mM Tris base buffer. In inhibition studies, test substances were applied from the apical side and were present during the incubation period only. During preincubation and incubation, the cells were continuously shaken and maintained at 37°C. 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 500 µL of 0.1% v/v Triton X-100 (dissolved in 5 mM Tris-HCl, pH 7.4). Radioactivity was measured by liquid scintilation counting.
Fractional outflow of intracellular [14C]-L-DOPA was evaluated in cells loaded with 2.5 µM [14C] L-DOPA for 6 min, then the corresponding efflux monitored over 24 min in the absence and the presence of different amino acids. Fractional outflow was calculated using the expression
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Cell viability
Cells were preincubated for 30 min at 37°C, then incubated in the absence or presence of L-DOPA and test compounds for 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, >95% of the cells excluded the dye.
Data analysis
Km and Vmax values for the uptake of [14C] L-DOPA, as determined from a competitive uptake inhibition protocol (23)
, were calculated from nonlinear regression analysis using the GraphPad Prism statistics software package (24)
. Arithmetic means are given with SE. Statistical analysis was performed by one-way ANOVA, followed by Newman-Keuls test for multiple comparisons. A P value of <0.05 was assumed to denote a significant difference.
Drugs
L- and D-amino acids, 2-aminobicyclo (2, 2, 1)-heptane-2-carboxylic acid (BCH), N-(methylamino)-isobutyric acid, and Trypan blue were purchased from Sigma. Tolcapone was kindly donated by late Professor Mosé Da Prada (Hoffman La Roche, Basle, Switzerland). [14C] L-DOPA, specific activity 51 mCi/mmol, was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The predicted amino acid sequence of this LLC-PK1 LAT2 cDNA fragment codes for a protein fragment of 392 amino acid residues covering residues 1-397 of the human LAT2 protein. This LLC-PK1 LAT2 protein fragment shows an amino acid sequence identity of 94% with its human, rabbit, rat, and mouse counterparts and 91% identity with opossum LAT2. The presence of LAT1 and LAT2 transcripts in pig renal LLC-PK1 cells was then examined by RT-PCR using specific primers for LAT1 or LAT2 Sus scrofa cDNA. The products obtained had the expected size, shown in Fig. 2
A: a 1029 bp fragment corresponding to LAT1 and a 437 bp fragment corresponding to LAT2.
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One target region of pig LAT2 mRNA was selected by scanning the length of the LAT2 gene for AA-dinucleotide sequences and downstream 19 nucleotides without significant homology to other genes by using an appropriate genome database. The antisense strand of synthesized LAT2-siRNA is the reverse complement of the target sequences (Table 1)
. The sense strand of the LAT2-siRNA has the same sequence as the target mRNA sequence, except it lacks the 5'-AA sequence (Table 1)
. A uridine dimer was incorporated at the 3' end of the sense strands siRNAs (Table 1)
. Thus, the end product is a double-stranded 21-mer siRNA that, theoretically, should reduce the expression of LAT2 mRNA and protein and a mismatch siRNA that should not be effective in LAT2 gene silencing.
In the next series of experiments, LLC-PK1 cells were treated with a siRNA targeted against the sequence corresponding to nt 97-117 (AAG AAA GAG ATT GGA TTG GTC) or a mismatch sequence (AAG AAA GAG GAG TAT TTG GTC). The dsRNAs were complexed with lipofectin (3%) and added to the cells for 12 h in serum-free medium. Levels of LAT2 cDNA, as determined by real-time quantitative RT-PCR, in untreated LLC-PK1 cells were 3.4 ± 0.4 fg cDNA/50 ng total RNA, a significant reduction 24 h after transfection with the LAT2 siRNA (25 nM) targeted against the sequence corresponding to nt 97-117 of the pig LAT2 gene (Fig. 2B
). Levels of LAT2 cDNA in cells treated with lipofectin (3%) and the mismatch LAT2 siRNA (25 nM) did not differ from those in untreated LLC-PK1 cells (Fig. 2B
). The presence of LAT1 protein in LLC-PK1 cells was studied by immunoblotting using an antibody raised against the rat LAT1. As shown in Fig. 2C
, the antibody against LAT1 recognized the presence in LLC-PK1 cells of a protein of the expected size (40 kDa), the abundance of which was similar in cells transfected with siRNAs against the native target sequence (siRNA LAT2) and the mismatch target sequence (mismatch siRNA LAT2).
In experiments designed to determine the kinetics of L-DOPA uptake transporter, cells were incubated for 6 min, when uptake was linear (14)
, with [14C]-L-DOPA (0.25 µM) in the absence or presence of increasing concentrations of unlabeled L-DOPA (Fig. 3
A). Km and Vmax of [14C]-L-DOPA uptake, determined by nonlinear analysis of the inhibition curve by unlabeled L-DOPA, were 120.0 ± 12.7 (µM) and 5187 ± 717 (pmol/mg protein/6 min). Substrate selectivity of L-DOPA uptake was evaluated in inhibition experiments in which 0.25 µM [14C]-L-DOPA uptake was measured in the presence of 1 mM of unlabeled L-amino acids (Fig. 3B
). Accumulation of [14C]-L-DOPA in LLC-PK1 cells was inhibited by L-isomers of the small and large neutral amino acids (alanine, serine, threonine, cysteine, leucine, isoleucine, phenylalanine, methionine, and tyrosine), histidine, tryptophan, valine, asparagine, and glutamine. Glycine, proline, basic amino acids arginine, lysine, and cystine, and acidic amino acids aspartate and glutamate did not inhibit uptake of [14C]-L-DOPA (Fig. 3B
). The effect of the amino acid analogs N-(methylamino)-isobutyric acid (MeAIB) and BCH, inhibitors of the A- and L-type amino acid transporters, respectively, was also evaluated. As depicted in Fig. 3B
, BCH but not MeAIB produced a marked decrease in [14C]-L-DOPA accumulation. The inhibitory effect of D-amino acids on [14C]-L-DOPA uptake was less marked than that obtained with L-isomers (data not shown). Irrespective of their optical conformation, the most effective neutral amino acids in reducing the uptake of [14C]-L-DOPA were leucine, isoleucine, phenylalanine, methionine, tyrosine, and tryptophan. On the other hand, D-DOPA failed to affect the accumulation of [14C]-L-DOPA.
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The efflux of L-DOPA was monitored over 24 min after loading the cells with [14C]-L-DOPA for 6 min. The efflux of [14C]-L-DOPA from LLC-PK1 cells over 24 min corresponded to 50% of the amount of the substrate accumulated in the cells. The intracellular levels of [14C]-L-DOPA remained almost unchanged in the first 6 min of the experiment, then progressively decreased until the end of the experiment (Fig. 4
A). The levels of [14C]-L-DOPA in the extracellular fluid mirrored those in the intracellular compartment (Fig. 4A
). The fractional outflow of [14C]-L-DOPA rose steadily for up to t = 24 min (Fig. 4A
). The efflux of [14C]-L-DOPA was also monitored in the absence and the presence of L-DOPA, L-leucine, L-arginine, or BCH (all at 1 mM). As shown in Fig. 4B
, at t = 9 min L-DOPA was slightly more potent than L-leucine in stimulating the efflux of [14C]-L-DOPA. BCH stimulated the efflux of [14C]-L-DOPA in LLC-PK1 cells, with a potency similar to that of L-DOPA. On the other hand, L-arginine did not stimulate the efflux [14C]-L-DOPA (Fig. 4B
). When cells loaded with [14C]-L-DOPA were incubated for 9 min with increasing concentrations of unlabeled L-DOPA, the efflux of [14C]-L-DOPA increased in a concentration-dependent manner (Fig. 4C
). Altogether, this would agree with the view that the L-DOPA transporter functions as an exchanger.
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All the inward and outward experiments mentioned above were performed in the presence of 140 mM Na+ in the uptake solution. Because amino acid transport across plasma membranes can be mediated by both Na+-dependent and -independent transporters, NaCl was replaced by an equimolar concentration of choline chloride in order to determine a potential Na+ dependency in [14C]-L-DOPA apical inward. As show in Table 2
, the effect of removing Na+ from the uptake solution produced a slight, but statistically significant (P<0.05), reduction of [14C] L-DOPA uptake. On the other hand, acidification of the uptake solution significantly increased the accumulation of [14C]-L-DOPA (Table 2)
. The spontaneous and L-DOPA-stimulated efflux of [14C]-L-DOPA was Na+ independent, but L-DOPA-stimulated efflux of [14C]-L-DOPA was markedly enhanced by the decrease in extracellular pH (Table 3
). Altogether, the results on the inward and outward transport of L-DOPA in LLC-PK1 cells indicate this may be promoted largely through the BCH-sensitive, Na+-independent, and pH-sensitive L-type amino acid transporter. A transporter with this type of characteristics most likely corresponds to LAT2, as LAT1 is characterized for being pH insensitive (11)
.
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The effect of LAT2 gene silencing on [14C]-L-DOPA uptake was evaluated 24 or 72 h after transfection with LAT2 siRNA; cells were then incubated with 0.25 µM [14C]-L-DOPA for 6 min. Incubation was performed in the absence of Na+ (NaCl was replaced by an equimolar concentration of choline chloride) at pH = 7.4. As shown in Fig. 5
A, 24 h after transfection with LAT2 siRNA [14C]-L-DOPA accumulation was decreased in a concentration-dependent manner not observed in cells treated with the mismatch LAT2 siRNA. As shown in Fig. 5B
, the decrease in [14C]-L-DOPA accumulation was a time-dependent effect, being more marked 72 h than 24 h after transfection with LAT2 siRNA. In experiments aimed to evaluate the effect of gene silencing on [14C]-L-DOPA outward transfer, cells were transfected with the LAT2 siRNA (25 nM) 72 h before the experiments. Cells were loaded with [14C]-L-DOPA for 6 min, then the corresponding efflux was monitored over 9 min in the absence of Na+. As shown in Fig. 5C
, the accumulation and efflux of [14C]-L-DOPA were markedly reduced in LAT2 siRNA-treated cells without changes in the spontaneous fractional outflow of [14C]-L-DOPA (Fig. 5C
). As indicated in Fig. 6
, the L-DOPA-stimulated fractional outflow of [14C]-L-DOPA in LAT2 siRNA-treated cells was identical to that observed in vehicle-treated cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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System L transports neutral amino acids with high affinity (Km in the µM range) with no need for Na+ in the extracellular medium and shows a very high capacity for trans-stimulation (25)
. The fraction of [14C]-L-DOPA uptake that does not require Na+, but is inhibited by BCH and neutral amino acids, may correspond to LAT2. This is substantiated by the following observations: 1) it is selective for neutral amino acids (26)
; 2) it is relatively nonspecific, binding both small and large amino acids (26)
, unlike LAT1 (26
, 27)
; 3) it is stimulated by acid pH (12
, 28)
, unlike LAT1 (11)
; and 4) it functions as a tightly coupled exchanger (26)
. However, amino acid specificity and affinity are different for LAT1 than LAT2. LAT1 induces Na+-independent transport of large neutral amino acids, with Km values in the micromolar range. LAT2 transports small neutral amino acids such as L-alanine, L-glycine, L-cysteine (13)
, but with a lower affinity to substrate amino acids than that of LAT1 (12)
. These findings are consistent with Km values for L-DOPA in LLC-PK1 cells. Accordingly, LLC-PK1 cells may transport [14C]-L-DOPA through the pH-dependent LAT2 transporter. This is further supported by the finding that that cells treated with an siRNA targeted against the LAT2 sequence markedly attenuated the uptake of [14C]-L-DOPA in LLC-PK1 cells.
The results of [14C]-L-DOPA efflux studies in LLC-PK1 cells are quite valuable when defining the nature of transporters involved in handling these substrates. Measurements of [14C]-L-DOPA efflux in the presence of extracellular amino acids showed a consistent efflux considerably greater than that in amino acid-free conditions. This suggests that the L-DOPA transporter functions as an exchanger. In fact, systems LAT1 and LAT2 function as exchangers (12
, 29
, 30)
, and L-DOPA induced outward of [14C]-L-DOPA agree with the view that either transporter may participate in the exchange. The finding that the efflux of [14C]-L-DOPA was insensitive to L-arginine and Na+, but sensitive to BCH and L-leucine, reinforces this view. The outward transfer of [14C]-L-DOPA was also sensitive to acidification of the extracellular medium (from pH 7.4 to 6.2). This suggests that the outward transfer of [14C]-L-DOPA might be promoted through LAT2 but not LAT1. This is supported by the finding that cells treated with a siRNA targeted against the LAT2 sequence markedly attenuated the uptake of [14C]-L-DOPA in LLC-PK1 cells. This is a strong argument against the involvement of LAT1, whose expression is not affected by siRNA LAT2, in L-DOPA uptake, because this cell line appears to express LAT1 and LAT2 transporters. On the other hand, it is interesting to underline the observation that the apical membrane in renal LLC-PK1 cells is endowed with other transporters for the handling of L-DOPA, namely, P-glycoprotein (14
, 31
32
33)
.
Though most [14C]-L-DOPA was entering the cells in a Na+-independent manner, a minor component of [14C]-L-DOPA uptake (25%) was found to require extracellular Na+. [14C]-L-DOPA uptake in LLC-PK1 cells was sensitive to inhibition by BCH, but not to MeAIB, and sensitive to inhibition by neutral, but not acidic and basic, amino acids. In addition, [14C]-L-DOPA uptake in LLC-PK1 cells shows trans-stimulation by unlabeled and L-DOPA. Taken together, these findings agree with the view that L-DOPA may be transported by systems B0 (Na+-dependent) and L (Na+-independent). System B0 is a broad specificity amino acid transport system cotransporting neutral amino acids with Na+ into cells that accepts BCH, but not MeAIB (25)
. Uptake of [14C]-L-DOPA was inhibited by neutral amino acids such as phenylalanine, leucine, and tyrosine and blocked by BCH, but not by MeAIB and the acidic and basic amino acids. For this reason it is likely that system B0 rather than system B0,+ or system y+L might be responsible for the Na+-dependent uptake of [14C]-L-DOPA. This is in line with previous observations showing that L-DOPA uptake in human and rat kidney slices is partially dependent on extracellular Na+ and sensitive to ouabain (34
35
36)
.
From a conceptual point of view, the present study adds new evidence in three important sectors. First, it shows that LAT2-siRNA effectively, specifically, and rapidly down-regulate endogenously expressed LAT2, reinforcing the view that siRNA-based gene silencing is an adequate technology with which to achieve selective gene suppression. Second, it demonstrates that LAT2 gene silencing markedly reduced the inward and outward transfer of [14C]-L-DOPA, suggesting a major role of LAT2 the regulation of the handling of L-DOPA at the kidney level. Third, it reveals functional characteristics of the mechanisms governing the availability of dopamine’s precursor, L-DOPA, at the renal level, where the amine plays the role of a local hormone regulating Na+ absorption.
In conclusion, it is likely that the sodium-independent uptake of L-DOPA include hetero amino acid exchanger LAT2, whose activation results in trans-stimulation of L-DOPA outward transfer. The predicted amino acid sequence of this LLC-PK1 LAT2 cDNA fragment codes for a protein fragment of 392 amino acid residues covering residues 1-397 of the human LAT2 protein. This LLC-PK1 LAT2 protein fragment shows an amino acid sequence identity of 94% with its human, rabbit, rat, and mouse counterparts. siRNA targeted against the sequence corresponding to nt 97-118 produced a concentration-dependent decrease in [14C]-L-DOPA accumulation that was not observed in cells treated with the mismatch sequence.
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ACKNOWLEDGMENTS |
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Received for publication April 15, 2004. Accepted for publication June 4, 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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