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【摘要】 The present study examined the functional characteristics of the inward and outward L -[ 14 C]DOPA and L -[ 14 C]leucine transporters in LLC-PK 1 cells. Uptake was initiated by the addition of Hanks' medium with a given concentration of L -[ 14 C]DOPA or L -[ 14 C]leucine. Saturation experiments were performed in cells incubated for 6 min with 0.25 µM concentration of the substrates in the absence and the presence of increasing concentrations of the nonlabeled substrates. Fractional outflow of intracellular L -[ 14 C]DOPA or L -[ 14 C]leucine was evaluated in cells loaded with 2.5 µM L -[ 14 C]DOPA or 1 µM L -[ 14 C]leucine for 6 min and then the corresponding efflux was monitored over 24 min. The high-affinity ( K m = 5.1 µM) uptake of L -[ 14 C]leucine and the low-affinity ( K m = 120.0 µM) uptake of L -[ 14 C]DOPA were largely promoted through a Na + -independent transporter. The uptake of the substrates was insensitive to N -(methylamino)-isobutyric acid but competitively inhibited by 2-aminobicyclo( 2, 2, 1 )-heptane-2-carboxylic acid (BCH). L - And D -neutral amino acids, but not acidic and basic amino acids, markedly inhibited L -[ 14 C]DOPA and L -[ 14 C]leucine accumulation. The uptake of L -[ 14 C]leucine was a pH-insensitive process, whereas that of L -[ 14 C]DOPA was sensitive to pH. The efflux of L -[ 14 C]DOPA and L -[ 14 C]leucine was markedly increased ( P < 0.05) by L -cysteine, L -leucine, BCH, and L -DOPA but not by L -arginine. RT-PCR detected LAT1 and LAT2 transcripts in LLC-PK 1 cells. It is concluded that LLC-PK 1 cells express both LAT1 and LAT2 transcripts and transport L -[ 14 C]leucine through the Na + -independent pH-insensitive and high-affinity LAT1 transporter, whereas L -[ 14 C]DOPA is mainly transported through the Na + -independent pH-insensitive and low-affinity LAT2 transporter and a minor component through a Na + -dependent transporter.
【关键词】 renal dopaminergic system sodium intake
THE RENAL DOPAMINERGIC SYSTEM is a local nonneuronal system constituted by epithelial cells of proximal convoluted renal tubules rich in aromatic L -amino acid decarboxylase (AADC) activity and using circulating or filtered L -DOPA as a source for dopamine ( 1, 22, 34 ). Dopamine, produced in proximal convoluted renal tubules, is in close proximity to proximal tubular epithelial cells that contain receptors for the amine and act as a paracrine or autocrine substance ( 33 ). The role of dopamine in the excretion of sodium after increased sodium intake led to the hypothesis that an aberrant renal dopaminergic system is important in the pathogenesis of some forms of genetic hypertension ( 15, 17, 19 ).
Although the kidney is endowed with one of the highest levels of AADC in the body and plasma levels of L -DOPA are in the nanomoles per milliliter range ( 13, 38 ), the rate-limiting step for the synthesis of dopamine in renal tissues is still a matter of debate. In fact, large amounts of taken-up L -DOPA in the kidney are not converted to dopamine ( 22, 34 ), suggesting that uptake of L -DOPA rather than its conversion to dopamine may rate limit the formation of renal dopamine. Recent studies from our laboratory showed that L -DOPA uptake in renal epithelial cells may be promoted through the L-type amino acid transporter, as has been found in intestinal epithelial cells ( 8 ) and at the level of brain capillary endothelium ( 2, 10, 11, 20, 31, 47 ). In this respect, it is interesting to underline the observation that the apical membrane in renal LLC-PK 1 cells is endowed with different transporters for the handling of L -DOPA (39-42). L -DOPA that is transported from the extracellular fluid to the intracellular space across the apical membrane occurs via the L-type amino acid transporter, whereas L -DOPA that is transported from the intracellular space to the extracellular fluid across the membrane occurs via a DIDS-sensitive mechanism ( 39 ). At present, candidate transport systems for apical-to-basal L -DOPA transfer may include the Na + -dependent systems B, B 0,+, and y + L and the Na + -independent systems L (LAT1 and LAT2) and b 0,+. Both b 0,+ and LAT1 were found to transport L -DOPA, the former in Xenopus laevis oocytes injected with poly A + RNA prepared from rabbit intestinal epithelium ( 16 ) and the latter in mouse brain capillary endothelial cells ( 20 ). L -DOPA in OK LC and OK HC cells is transported quite efficiently across the apical cell border and several findings indicate that L -DOPA uses at least two major transporters, systems LAT2 and b 0,+. The transport of L -DOPA by LAT2 corresponds to a Na + -independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by 2-aminobicyclo ( 2, 2, 1 )-heptane-2-carboxylic acid (BCH). The transport of L -DOPA by system b 0,+ is a Na + -independent transporter for neutral and basic amino acids that also recognizes the diamino acid cystine. Transporters involved in Na + -independent uptake of L -DOPA (systems LAT2 and b 0,+ ) also function as tightly coupled exchangers. In line with this view are recent data from our laboratory showing that overexpression of LAT2 in the spontaneously hypertensive rat (SHR) kidney is organ specific and precedes the onset of hypertension that is accompanied by enhanced ability to take up L -DOPA ( 28 ). This suggests that overexpression of renal LAT2 may constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natiuresis generally observed in this genetic model of hypertension ( 18 ). This adaptive mechanism may be limited to renal tissues, because at the intestinal level defective transduction of the D 1 receptor signal also occurs ( 23 ), but is not accompanied by increases in either dopamine tissue levels or intestinal LAT2 expression ( 28 ).
The present work aimed to evaluate the presence and define the kinetic characteristics of the LAT1 and LAT2 amino acid transporters in LLC-PK 1 cells, an established epithelial cell line derived from porcine renal tubule epithelial cells that retain several properties of proximal tubular epithelial cells in culture ( 14 ). For this purpose, we measured the activity of the apical inward and apical outward transport of L -[ 14 C]DOPA and L -[ 14 C]leucine in cell monolayers and evaluated the presence of LAT1 and LAT2 transcripts by RT-PCR. It is reported that LLC-PK 1 cells express both LAT1 and LAT2 transcripts and transport L -[ 14 C]leucine through the Na + -independent pH-insensitive and high-affinity LAT1 transporter, whereas L -[ 14 C]DOPA is mainly transported through the Na + -independent pH-insensitive and low-affinity LAT2 transporter and minor component through a Na + -dependent transporter.
METHODS
Cell culture. LLC-PK 1 cells were obtained from the American Type Culture Collection (Rockville, MD). LLC-PK 1 cells (ATCC CRL 1392; passages 198 - 206 ) were maintained in a humidified atmosphere of 5% CO 2 -95% air at 37°C and grown in medium 199 (Sigma, St. Louis, MO) supplemented with 100 U/ml of penicillin G, 0.25 µg/ml of amphotericin B, 100 µg/ml of streptomycin (Sigma), 3% fetal bovine serum (Sigma), and 25 mM HEPES (Sigma). For subculturing, the cells were dissociated with 0.05% trypsin-EDTA, split 1:4, and subcultured in Costar flasks with 75- or 162-cm 2 growth areas (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. The cell medium was changed every 2 days, and the cells reached confluence after 3-5 days of incubation. For 24 h before each experiment, the cell medium was free of fetal bovine serum. Experiments were generally performed 2-3 days after cells reached confluence and 6-8 days after the initial seeding, and each squared centimeter contained 80 µg of cell protein.
Transport of L -[ 14 C]DOPA and L -[ 14 C]leucine. On the day of the experiment, the growth medium was aspirated and 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 MgSO 4, 0.33 Na 2 HPO 4, 0.44 KH 2 PO 4, 0.25 CaCl 2, 1.0 MgCl 2, 0.15 Tris·HCl, and 1.0 sodium butyrate, pH 7.4. The incubation medium also contained benserazide (30 µM) and tolcapone (1 µM) to inhibit the enzymes AADC and catechol- O -methyltransferase, respectively. Apical uptake was initiated by the addition of 1 ml of Hanks' medium with a given concentration of the substrate. Time course studies were performed in experiments in which cells were incubated with 2.5 µM L -[ 14 C]DOPA or 2.5 µM L -[ 14 C]leucine for 1, 3, 6, 12, 30, and 60 min. Saturation experiments were performed in cells incubated for 6 min with 0.25 µM L -[ 14 C]DOPA or 0.25 µM L -[ 14 C]leucine in the absence and presence of increasing concentrations of the unlabeled substrate. In one set of experiments, L -[ 14 C]leucine accumulation was compared in (aminooxy)acetic acid-treated and nontreated cells to evaluate the contribution of transport and metabolism to L -[ 14 C]leucine accumulation ( 24 ). The transaminase inhibitor (aminooxy)acetic acid (2.5 mM) was added to the culture medium 15 min before the beginning of the transport experiment. The uptake of L -[ 14 C]leucine was then measured after 6 min, as described above. 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% vol/vol Triton X-100 (dissolved in 5 mM Tris·HCl, pH 7.4) to the cells. Radioactivity was measured by liquid scintillation counting.
Fractional outflow of intracellular L -[ 14 C]DOPA and L -[ 14 C]leucine was evaluated in cells loaded with 2.5 µM L -[ 14 C]DOPA or 1 µM L -[ 14 C]leucine for 6 min and then the corresponding efflux was monitored over 24 min, in the absence and presence of different amino acids. Fractional outflow was calculated using the expression
where a.a fluid [ 14 C] L - indicates the amount of radiolabeled amino acid (in pmol/mg protein) that reached the fluid bathing the apical cell side and a.a cell [ 14 C] L - (in pmol/mg protein) indicates the amount of radiolabeled amino acid accumulated in the cell monolayer. The protein content of cell monolayers was determined using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard.
RT-PCR. Total RNA was isolated from cell monolayers using TRIzol (Invitrogen) according to the manufacturer's instructions. The RNA obtained was dissolved in diethylpyrocarbonate-treated water and quantified by spectrophotometry at 260 nm. Five micrograms of 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 degenerated set simultaneously specific for human, rat, mice, and pig 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 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 Taq PCRx DNA Polymerase (Invitrogen) with 1 x enhancer, for both 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.
Cell viability. Cells were preincubated for 30 min at 37°C and then incubated in the absence or presence of L -DOPA and test compounds for 6 min further. 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 using a Leica microscope. Under these conditions, more than 95% of the cells excluded the dye.
Data analysis. K m and V max values for the uptake of L -[ 14 C]DOPA and L -[ 14 C]leucine, as determined from a competitive uptake inhibition protocol ( 6 ), were calculated from nonlinear regression analysis using the GraphPad Prism statistics software package ( 25 ). The following variables concerning L -[ 14 C]DOPA and L -[ 14 C]leucine fractional outflow were derived from the individual fractional outflow profiles: maximum efflux (E max ), time to attain the half-maximal efflux ( t half-Emax ), and area under the effect-time curve (AUEC). The variables t half-Emax and AUEC were calculated from nonlinear regression analysis using the GraphPad Prism statistics software package ( 25 ). Arithmetic means are given with SE. Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls test for multiple comparisons. A P value <0.05 was assumed to denote a significant difference.
Drugs. L - And D -amino acids, BCH, N -(methylamino)-isobutyric acid, and Trypan blue were purchased from Sigma. Tolcapone was kindly donated by the late Professor Mosé Da Prada (Hoffman La Roche, Basel, Switzerland). L -[ 14 C]leucine-specific activity of 303 mCi/mmol and L -[ 14 C]DOPA-specific activity of 51 mCi/mmol were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK).
RESULTS
Previous studies showed that uptake of nonsaturating concentrations (2.5 µM) of L -DOPA in LLC-PK 1 cells was linear with time for up to 12 min of incubation ( 41 ). The accumulation of L -[ 14 C]leucine (in pmol·mg protein -1 ·6 min -1 ) was not affected by pretreatment with the transamination inhibitor (aminooxy)acetic acid (data not shown). It was, therefore, decided to examine the time-dependent accumulation of both L -[ 14 C]DOPA and L -[ 14 C]leucine at 2.5 µM. As shown in Fig. 1 A, both L -[ 14 C]DOPA and L -[ 14 C]leucine were rapidly accumulated in LLC-PK 1 cells. However, for L -[ 14 C]DOPA equilibrium was attained at 30 min of incubation, whereas for L -[ 14 C]leucine uptake was linear up to 60 min. At 6 min, when uptake was linear for both substrates and considering intracellular water as 7.0 ± 0.7 µl/mg protein ( 41 ), the intracellular concentration of L -[ 14 C]DOPA and L -[ 14 C]leucine was, respectively, 9.0 ± 0.5 and 14.5 ± 1.4 µM at a medium concentration of 2.5 µM. This represented a cell-to-medium concentration ratio of 3.6 for L -[ 14 C]DOPA and 5.8 for L -[ 14 C]leucine. In a subsequent set of experiments designed to determine the kinetics of the L-type amino acid transporter, cells were incubated for 6 min with L -[ 14 C]DOPA or L -[ 14 C]leucine, both at 0.25 µM, in the absence or presence of increasing concentrations of unlabeled L -DOPA and L -leucine ( Fig. 1, B and C ). Kinetic parameters of L -[ 14 C]DOPA and L -[ 14 C]leucine uptake ( K m and V max ) were determined by nonlinear analysis of the inhibition curve for L -DOPA and L -leucine and are given in Table 1. The affinity of the transporter for -[ 14 C]leucine, as evidenced by K m values, was 20 times greater ( P < 0.05) than that for -[ 14 C]DOPA ( Table 1 ). On the other hand, it is interesting to note that the affinity of the L -leucine transporter for L -DOPA was similar to that for L -DOPA. This is particularly evidenced by the finding that inhibition of -[ 14 C]DOPA by unlabeled L -DOPA was similar to the inhibition of -[ 14 C]leucine uptake by unlabeled L -DOPA ( Table 1 ). By contrast, unlabeled L -leucine was more potent in inhibiting -[ 14 C]leucine uptake than in inhibiting -[ 14 C]DOPA uptake ( Table 1 ), which suggests that the affinity of the L -DOPA transporter for L -leucine is lower than that of the L -leucine transporter.
Fig. 1. A : time course of L -[ 14 C]DOPA and L -[ 14 C]leucine accumulation in LLC-PK 1 cells. Cells were incubated at 37°C for 1, 3, 6, 12, 30, and 60 min in the presence of L -[ 14 C]DOPA (2.5 µM) or L -[ 14 C]leucine (2.5 µM). Inset : accumulation L -[ 14 C]DOPA in LLC-PK 1 cells. Effect of increasing medium concentrations of L -DOPA and L -leucine (0.3 to 3,000 µM) on the uptake of L -[ 14 C]DOPA (0.25 µM; B ) and L -[ 14 C]leucine (0.25 µM; C ) in LLC-PK 1 cells is shown. Each symbol represents the means ± SE, n = 4-8.
Table 1. K m and V max values for the saturable component of -[ 14 C]DOPA and -[ 14 C]leucine uptake and IC 50 values for the inhibition of -[ 14 C]DOPA and -[ 14 C]leucine uptake in cultured LLC-PK 1 cells by unlabeled L -DOPA and L -leucine
Substrate selectivity of L -DOPA and L -leucine uptake was studied in inhibition experiments in which 0.25 µM -[ 14 C]DOPA or 0.25 µM -[ 14 C]leucine uptake was measured in the presence of 1 mM unlabeled L -amino acids ( Table 2 ); this concentration is approximately four- to eightfold the physiological plasma levels ( 4, 5 ). The accumulation of -[ 14 C]DOPA and -[ 14 C]leucine in LLC-PK 1 cells was inhibited by L -isomers of the neutral amino acids, histidine, valine, asparagine, and glutamine. Glycine, proline, the basic amino acids arginine, lysine, and cystine and the acidic amino acids aspartate and glutamate did not inhibit uptake of -[ 14 C]DOPA and -[ 14 C]leucine ( Table 2 ). The inhibitory effect of D -amino acids on -[ 14 C]DOPA and -[ 14 C]leucine uptake was less marked than that obtained with L -isomers, as shown in Table 2. Irrespective of their optical conformation, the most effective neutral amino acids in reducing the uptake of -[ 14 C]DOPA and -[ 14 C]leucine were leucine, isoleucine, phenylalanine, methionine, tyrosine, and tryptophan. On the other hand, D -DOPA failed to affect the accumulation of -[ 14 C]DOPA and -[ 14 C]leucine, whereas L -DOPA markedly inhibited -[ 14 C]DOPA and -[ 14 C]leucine uptake ( Table 2 ).
Table 2. Effect of the indicated L -amino acids (1 mM) and D -amino acids (1 mM) on the uptake of -[ 14 C]DOPA and -[ 14 C]leucine (0.25 µM) in LLC-PK 1 cells
All the 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 Na + -independent transporters, NaCl was replaced by an equimolar concentration of choline chloride to determine a potential Na + dependency in -[ 14 C]DOPA and -[ 14 C]leucine uptake. The effect of removing Na + from the uptake solution produced a slight, but statistically significant ( P < 0.05), reduction in -[ 14 C]DOPA accumulation ( Fig. 2 A ) but did not alter -[ 14 C]leucine accumulation ( Fig. 2 B ). Altogether, these results indicate that transport of L -DOPA and L -leucine in LLC-PK 1 cells may be largely promoted through the L-type amino acid transporter, although a minor component in L -DOPA uptake also involves a Na + -dependent transporter. To confirm this hypothesis, the next series of experiments was addressed to study 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. As depicted in Table 2, BCH, but not MeAIB, produced a marked decrease in -[ 14 C]DOPA and -[ 14 C]leucine accumulation. These results suggest that the inward transfer of L -DOPA and L -leucine in LLC-PK 1 cells may be largely promoted through the BCH-sensitive and Na + -independent L-type amino acid transporter. Apart from amino acid specificity and affinity profile, a major difference between LAT1 and LAT2 is that the former is pH insensitive ( 9, 29 ) and the latter is pH sensitive ( 8, 12, 32 ). The effect of pH on -[ 14 C]DOPA and -[ 14 C]leucine influx was examined by changing the pH of the uptake solution. -[ 14 C]DOPA transport was greater at an acidic pH ( Fig. 2 C ), whereas -[ 14 C]leucine accumulation was not affected by changing pH of the incubation medium ( Fig. 2 D ). In fact, the pH-sensitive L -DOPA uptake was linear from pH values of 7.4 to 5.0, being the rate of uptake 2.8 ± 0.2 pmol·mg protein -1 ·pH unit -1.
Fig. 2. Effect of sodium and pH on the intracellular accumulation of -[ 14 C]DOPA (0.25 µM) and -[ 14 C]leucine (0.25 µM) in monolayers of LLC-PK 1 cells. Each column or symbol represents the means ± SE, n = 8. * P < 0.05, significantly different from corresponding control values.
Because some amino acid transporters have been shown to function as amino acid exchangers, a new series of experiments was conducted in cells loaded with -[ 14 C]DOPA or -[ 14 C]leucine for 6 min and then the corresponding efflux was monitored over 24 min. The efflux of -[ 14 C]DOPA and -[ 14 C]leucine from LLC-PK 1 cells over 24 min corresponded to 75 and 50% of the amount of the corresponding substrates accumulated in the cells. The intracellular levels of -[ 14 C]DOPA remained almost unchanged in the first 6 min of the experiment and then progressively decreased until the end of the experiment ( Fig. 3 A ). As shown in Fig. 3 B, the intracellular levels of -[ 14 C]leucine markedly decreased in the first 9 min of the experiment and remained almost unchanged until the end of the collection period. The levels of -[ 14 C]DOPA and -[ 14 C]leucine in the extracellular fluid mirrored those in the intracellular compartment ( Fig. 3, A and B ). The fractional outflow of -[ 14 C]DOPA rose steadily up to t = 24 min ( Fig. 3 C ). The fractional outflow of -[ 14 C]leucine rose steadily up to t = 9 min, then remaining at the 50% level until the end of the experiment ( Fig. 3 C ). A more detailed analysis reveals that major differences between -[ 14 C]DOPA and -[ 14 C]leucine fractional outflow are concerned with E max and t half-Emax. In fact, E max values for spontaneous -[ 14 C]DOPA fractional outflow (67.5 ± 1.5%, n = 12) were significantly greater ( P < 0.05) than those for -[ 14 C]leucine (52.7 ± 1.6%, n = 12). On the other hand, t half-Emax values for -[ 14 C]leucine (3.4 ± 0.2 min, n = 12) were significantly ( P < 0.05) lower than those for -[ 14 C]DOPA (8.6 ± 0.4 min, n = 12). When cells loaded with -[ 14 C]DOPA or -[ 14 C]leucine were incubated for 9 min with increasing concentrations of unlabeled L -DOPA or unlabeled L -leucine, the efflux of -[ 14 C]DOPA and -[ 14 C]leucine increased in a concentration-dependent manner ( Fig. 4, A and B ). This is in agreement with the view that the L -DOPA transporter and the L -leucine transporter function as exchangers. Similar to that observed for the inward transport of -[ 14 C]DOPA, D -DOPA failed to affect the outward transfer of -[ 14 C]DOPA ( Fig. 4 C ). By contrast, the leucine-stimulated outward transfer of -[ 14 C]leucine was of similar magnitude when D -leucine was used ( Fig. 4 D ). In another series of experiments, the efflux of -[ 14 C]DOPA and -[ 14 C]leucine was monitored for 24 min in the absence and presence of L -cysteine, L -leucine, L -arginine, BCH, or L -DOPA, all at 1 mM ( Fig. 5 ). Table 3 depicts E max and t half-Emax values and percent change in the AUEC of -[ 14 C]DOPA and -[ 14 C]leucine fractional outflow in the absence and presence of the amino acids. As shown in Table 3, L -cysteine, L -leucine, BCH, and L -DOPA, but not L -arginine, significantly ( P < 0.05) increased E max and AUEC values of -[ 14 C]DOPA fractional outflow and markedly ( P < 0.05) reduced t half-Emax values (see also Fig. 5 A ). The efflux of -[ 14 C]leucine was also markedly stimulated by L -cysteine, L -leucine, BCH, and L -DOPA but not by L -arginine ( Fig. 5 B ). In contrast to that observed for -[ 14 C]DOPA fractional outflow, L -cysteine failed to increase E max values for -[ 14 C]leucine fractional outflow, despite increases in AUEC and decreases in t half-Emax values ( Table 3 ). The next series of experiments evaluated the Na + dependence and pH sensitivity of -[ 14 C]DOPA and of -[ 14 C]leucine fractional outflow. As shown in Fig. 6, A and B, the L -DOPA- and L -leucine-stimulated efflux of, respectively, -[ 14 C]DOPA and -[ 14 C]leucine was Na + independent. The L -DOPA-stimulated efflux of -[ 14 C]DOPA was significantly enhanced by the decrease of extracellular pH ( Fig. 6 C ). By contrast, extracellular acidification failed to alter the L -leucine-stimulated outward transfer of -[ 14 C]leucine ( Fig. 6 D ).
Fig. 3. Levels of -[ 14 C]DOPA and -[ 14 C]leucine in cells ( A ) and incubation fluid ( B ) and fractional outflow (%; C ) in LLC-PK 1 cells. Cells were loaded for 6 min with 2.5 µM -[ 14 C]DOPA or 1.0 µM -[ 14 C]leucine and then incubated in 24 min. Each symbol represents the means ± SE, n = 12.
Fig. 4. A and B : fractional outflow (%) of -[ 14 C]DOPA and -[ 14 C]leucine ( L -Leu) in LLC-PK 1 cells in the absence (control) and presence of L -DOPA and L -Leu. Cells were loaded for 6 min with 2.5 µM -[ 14 C]DOPA or 1 µM [ 14 C] -Leu and then incubated in the presence of increasing concentrations of unlabeled L -DOPA and L -Leu (1 mM) for 9 min. C and D : fractional outflow (%) of -[ 14 C]DOPA and [ 14 C] -Leu in the presence of L -DOPA (1 mM) and D -DOPA (1 mM) or L -Leu (1 mM) and D -Leu (1 mM). Cells were loaded for 6 min with 2.5 µM -[ 14 C]DOPA or 1 µM [ 14 C] -Leu and then incubated in the absence or presence of unlabeled L - or D -DOPA and L - or D -Leu for 9 min. Each symbol represents the means ± SE, n = 8. * P < 0.05, significantly different from corresponding control value.
Fig. 5. Fractional outflow (%) of -[ 14 C]DOPA ( A ) and [ 14 C] -Leu ( B ) in LLC-PK 1 cells in the absence (control) and presence of L -cysteine ( L -Cys), L -Leu, L -arginine ( L -Arg), 2-aminobicyclo ( 2, 2, 1 )-heptane-2-carboxylic acid (BCH), or L -DOPA. Cells were loaded for 6 min with 2.5 µM -[ 14 C]DOPA or 1 µM [ 14 C] -Leu and then incubated in the absence or presence of unlabeled L -Cys (1 mM), L -Leu (1 mM), L -Arg (1 mM), BCH (1 mM), or L -DOPA (1 mM) for 1, 3, 6, 9, 12, and 24 min. Each symbol represents the means ± SE, n = 4. * P < 0.05, significantly different from corresponding control value.
Table 3. E max, t half-E max, and AUEC of fractional outflow of -[ 14 C]DOPA and -[ 14 C]leucine in LLC-PK 1 cells
Fig. 6. Effect of sodium ( A and B ) and pH ( C and D ) on spontaneous and L -DOPA- or L -Leu-stimulated fractional outflow of -[ 14 C]DOPA or [ 14 C] -Leu in LLC-PK 1 cells. Cells were loaded for 6 min with 1 µM [ 14 C] -Leu and then incubated in the absence or presence of L -DOPA (1 mM) and L -Leu (1 mM) for 12 min. Each column represents the means ± SE, n = 4. * P < 0.05, significantly different from corresponding control value. # P < 0.05, significantly different from corresponding value for pH 7.4.
Altogether, the results on the inward and outward transport of L -DOPA in LLC-PK 1 cells indicate that this may be largely promoted 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 ( 8, 29 ). The presence of LAT1 and LAT2 transcripts in pig renal LLC-PK 1 cells was then examined by RT-PCR using specific primers for either LAT1 or LAT2 Sus scrofa cDNAs. The products obtained had the expected size as shown in Fig. 7 : a 1,029-bp fragment corresponding to LAT1 and a 437-bp fragment corresponding to LAT2.
Fig. 7. RT-PCR detection of LAT1 and LAT2 in total RNA extracted from LLC-PK 1 cells. MW, molecular weight with 250-bp DNA ladder.
Both LAT1 and LAT2 have been shown to be associated with the single transmembrane domain 4F2hc protein through an intermolecular disulfide bond ( 26, 43, 48 ). Hg 2+ and other organic mercury compounds have been demonstrated to inactivate the transporter by covalently modifying cysteine residues, this being reversed by the reducing agent -mercaptoethanol ( 7 ). As shown in Fig. 8, Hg 2+ was found to completely prevent the L -DOPA-stimulated outward transfer of -[ 14 C]DOPA in LLC-PK 1 cells, this being partially prevented by the reducing agent -mercaptoethanol. The L -leucine-stimulated outward transfer of -[ 14 C]leucine was also completely blocked by Hg 2+, and this was prevented by -mercaptoethanol ( Fig. 8 ).
Fig. 8. Spontaneous and -DOPA or L -Leu-stimulated fractional outflow of -[ 14 C]DOPA and [ 14 C] -Leu in cells treated with vehicle, mercury chloride (200 µM), and mercury chloride (200 µM) plus -mercaptoethanol (2 mM). Cells were loaded for 6 min with 2.5 µM -[ 14 C]DOPA or 1 µM [ 14 C] -Leu and then incubated in the absence or presence of L -DOPA (1 mM) or L -Leu (1 mM) for 12 min. Each column represents the means ± SE, n = 4. * P < 0.05, significantly different from corresponding control value.
DISCUSSION
The present study shows that LLC-PK 1 cells transport quite efficiently L -DOPA and L -leucine through the apical cell side, and several findings demonstrate that this uptake process is a facilitated mechanism. Although most of -[ 14 C]DOPA was entering the cells in a Na + -independent manner, a minor component of -[ 14 C]DOPA uptake ( 25%) was found to require extracellular Na +, which contrasts with the Na + -independent L -leucine apical transfer. This is in line with previous observations showing that L -DOPA uptake in human and rat kidney slices is a Na + -dependent and ouabain-sensitive process (35-37). Apart from this, -[ 14 C]DOPA and -[ 14 C]leucine uptake in LLC-PK 1 cells were both sensitive to inhibition by BCH, but not to MeAIB, and sensitive to inhibition by neutral but not acidic and basic amino acids. In addition, -[ 14 C]DOPA and -[ 14 C]leucine uptake in LLC-PK 1 cells shows trans -stimulation by unlabeled L -DOPA and L -leucine. Taken together, these findings agree with the view that L -DOPA may be transported by systems B 0 (Na + dependent) and L (Na + independent), whereas L -leucine may be transported through system L only. However, L -DOPA and L -leucine appear to be handled through system L in a very different manner, as L -DOPA is transported by a low-affinity and pH-sensitive process whereas L -leucine is transported by a high-affinity and pH-insensitive transport, which may correspond to LAT2 and LAT1 transporters, respectively.
System L transports neutral amino acids with high affinity ( K m in the µM range) with no need for Na + in the extracellular medium and shows very high capacity for trans -stimulation ( 26 ). The fraction of -[ 14 C]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 ( 27 ); 2 ) it is relatively nonspecific, binding both small and large amino acids ( 27 ), unlike LAT1 ( 21, 27 ); 3 ) it is stimulated by acid pH ( 4, 32 ), unlike LAT1 ( 27 ); and 4 ) it functions as a tightly coupled exchanger ( 27 ). On the other hand, the finding that accumulation of -[ 14 C]leucine in LLC-PK 1 cells was significantly insensitive to changes in extracellular pH fits well with the view that L -leucine uptake in these cells is promoted through LAT1 that is characterized for being pH insensitive ( 9, 29 ). However, amino acid specificity and affinity are different for LAT1 compared with LAT2. LAT1 induces Na + -independent transport of large neutral amino acids with K m values in the micromolar range. LAT2 also transports small neutral amino acids such as L -alanine, L -glycine, and L -cysteine ( 48 ), however, with a lower affinity to substrate amino acids than that of LAT1 ( 32 ). These findings are consistent with K m values for L -DOPA and L -leucine in LLC-PK 1 cells, as depicted in Fig. 1 and Table 1.
The results of -[ 14 C]DOPA and -[ 14 C]leucine efflux studies in LLC-PK 1 cells are also quite valuable to define the nature of transporters involved in the handling of these substrates. Measurements of -[ 14 C]DOPA and -[ 14 C]leucine efflux in the presence of extracellular amino acids did show a consistent efflux, which was considerably greater than that in amino acid-free conditions. This suggests that L -DOPA and L -leucine transporters function as exchangers. In fact, systems LAT1 and LAT2 function as exchangers ( 3, 30, 32 ) and L -DOPA- and leucine-induced outward transfer of both substrates agrees with the view that either transporter may participate in the exchange. The finding that the efflux of both -[ 14 C]DOPA and -[ 14 C]leucine was insensitive to L -arginine and Na +, but sensitive to L -cysteine, L -leucine, and BCH, reinforces this view. However, as mentioned for the inward transport of -[ 14 C]DOPA and -[ 14 C]leucine, it appears that the outward transport of -[ 14 C]DOPA and -[ 14 C]leucine is promoted through systems with slightly different characteristics. The outward transfer of -[ 14 C]DOPA was sensitive to acidification of the extracellular medium (from pH 7.4 to 6.2), whereas the outward transfer of -[ 14 C]leucine was not sensitive to extracellular acidification. This suggests that the outward transfer of -[ 14 C]DOPA and -[ 14 C]leucine might be promoted through LAT2 and LAT1, respectively. This is in line with the finding that LLC-PK 1 cells express both LAT1 and LAT2, as evidenced by the presence of LAT1 and LAT2 transcripts. The finding that Hg 2+ completely prevented the stimulated outward transfer of -[ 14 C]DOPA and -[ 14 C]leucine, this being prevented by -mercaptoethanol, strongly suggests that in LLC-PK 1 cells LAT1 and LAT2 might be associated with the single transmembrane domain 4F2hc protein through an intermolecular disulfide bond.
From a conceptual point of view, the present study adds new evidence in three important sectors. First, it reveals the 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. Second, it shows that neutral amino acids may be of importance for the regulation of the inward/outward transfer of L -DOPA, which is in line with the view that high-protein intake largely affects the renal synthesis of dopamine. Third, despite the fact that high-salt intake ( 23, 44 - 46 ) also increases the renal synthesis of dopamine, the Na + -dependent L -DOPA uptake may be of limited importance under in vivo conditions given the narrow oscillations of extracellular Na +. Finally, the pH sensitivity of L -DOPA uptake may be also of limited importance given the modest effect within the pH range compatible with life.
It is concluded that LLC-PK 1 cells express both LAT1 and LAT2 transcripts and transport -[ 14 C]leucine through the Na + -independent pH-insensitive and high-affinity LAT1 transporter, whereas -[ 14 C]DOPA is mainly transported through the Na + -independent pH-insensitive and low-affinity LAT2 transporter and a minor component through a Na + -dependent transporter.
GRANTS
This work was supported by Grant POCTI/CBO/42788/2001 from Fundação para a Ciência e a Tecnologia.
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作者单位：Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal