This study examined the inward transport of l-[14C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[14C]alanine uptake was highly dependent on extracellular Na+. Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na+-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na+-dependent l-alanine transport under Vmax conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar Km values. Differences in magnitude of Na+-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na+-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.
- l-alanine transport
- alanine-serine-cysteine-threonine transporter-2
transport of neutral amino acids across membranes of mammalian cells proceeds through a variety of different transport systems (reviewed in Refs. 4, 9, and 18). At the level of the kidney and small intestine epithelia, distinct transporters are located in the apical and basolateral membranes to ensure the vectorial transport of amino acids across the epithelial cells (13). Recently, several amino acid transporters have been identified and shown to play a role in the cellular uptake and/or basolateral extrusion of neutral amino acids. Indeed, it has been proposed (13) that neutral amino acids are absorbed from the luminal fluid via Na+-dependent systems, like the proline transporter IMINO/SIT (SLC6A20) (20, 37), the neutral amino acid exchanger ASCT2 (SLC1A5) (16), or the broad specific neutral amino acid transporter B0AT1 (SLC6A19), whose molecular structure has been identified recently (7). The exit path for neutral amino acids to the blood stream is supposed to proceed through the system L. In kidney and small intestine epithelial cells, type-2 l-amino acid transport (LAT2), together with 4F2hc, was found to be present in basolateral membrane, which is well suited for the exit path of intracellular amino acids (28, 32). System y+L, most likely y+LAT1 (38, 45), located at the basolateral membrane, is expected to mediate the obligatory exchange of intracellular basic amino acids against extracellular neutral amino acids, cotransported with sodium ions (27). Because these two transport systems function as obligatory exchangers, they cannot contribute to the net transepithelial transport of amino acids but are thought to play a role in extending the transport selectivity of putative parallel functioning by unidirectional transporters.
System ASC transport activity is ubiquitous and characterized by its preference for small neutral amino acids including alanine, serine, and cysteine. The system ASC of neutral amino acid transporters (SLC1A4 and SLC1A5) belongs to the solute carrier family-1 (SLC1), which also includes the high-affinity glutamate transporters (13, 14, 40, 46). Human ATB0 was identified by RT-PCR and enzymatic restriction analysis in the human proximal tubule cell line HKPT (17) and corresponds to rodent ASCT2. The two ASC transporters exhibit distinct substrate selectivity. SLC1A4 encodes the Na+-dependent amino acid transporter ASCT1, which accepts l-alanine, l-serine, l-theonine, and l-cysteine in a stereospecific manner. ASCT2, the second isoform of the ASC transport system, is encoded by SLC1A5. In the kidney and intestine, ASCT2 is present in the brush-border membranes of the proximal tubule cells and enterocytes, respectively (3). In addition to the typical system ASC substrates, it also accepts l-glutamine and l-asparagine at higher affinity as well as methionine, leucine, and glycine with lower affinity. Both ASCT1 and ASCT2 mediate the Na+-dependent obligatory exchange of substrate amino acids (5, 38, 46).
We previously reported that overexpression of Na+-independent LAT2 in the spontaneously hypertensive rat (SHR) kidney is organ specific and precedes the onset of hypertension. This overexpression is accompanied by an enhanced ability to take up l-3,4-dihydroxyphenylalanine (l-DOPA) (29). These observations formed the basis for the hypothesis that overexpression of renal LAT2 leads to enhanced renal production of dopamine in the SHR in an attempt to compensate for the decreased dopamine-mediated natriuresis generally observed in this genetic model of hypertension. Furthermore, we have demonstrated that immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto rats (WKY) and SHR transport l-DOPA quite efficiently through the apical cell border, in a Na+-independent manner (30). LAT2 was almost exclusively responsible for l-DOPA transport in WKY cells, whereas in SHR cells, 25% of l-DOPA uptake was through a Na+-dependent system, 25% through LAT2, and the remaining 50% through LAT1. Differences in l-DOPA handling between SHR and WKY cells may result from the overexpression of LAT1 and LAT2 transporters in the former (30).
In an attempt to understand better differences in the handling of l-amino acids in hypertension, the present study examined the function and expression of ASCT2. The inward transport of l-[14C]alanine, an ASCT2 preferential substrate, was evaluated in monolayers of immortalized renal PTE cells from the SHR and its normotensive control, WKY. The quantification of ASCT2 mRNA and ASCT2 protein was performed in immortalized renal PTE cells and kidney cortices from WKY and SHR.
METHODS AND MATERIALS
Immortalized renal PTE cells from WKY and SHR (44) were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. SHR and WKY PTE cells were grown in DMEM Nutrient Mixture-Ham's F-12 (Sigma, St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 μg/ml amphotericin B, 100 μg/ml streptomycin (Sigma), 5% fetal bovine serum (Sigma), and 25 mM HEPES (Sigma). For subculturing, the cells were dissociated with 0.10% trypsin-EDTA, split 1:4, and subcultured in Costar flasks with 75- or 162-cm2 growth areas (Costar, Badhoevedorp, The Netherlands). 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 cells were maintained in fetal bovine serum-free medium. Experiments were generally performed 2–3 days after cells reached confluence and 6–8 days after the initial seeding; each squared centimeter contained ∼80–100 μg of cell protein.
Uptake of l-amino acids.
Flux measurements in immortalized renal PTE cells from the WKY and SHR were performed as previously described (30). Briefly, on the day of the experiment, growth medium was aspirated, and the cell monolayers were preincubated for 15 min in Hanks' medium at 37°C. The Hanks' medium had the following composition (in 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. 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 0.25 μM l-[14C]alanine for 1, 3, 6, 12, 30, and 60 min. Saturation experiments were performed in cells incubated for 6 min with 0.25 μM radiolabeled amino acid in the absence and in the presence of increasing concentrations of the unlabeled substrate. To achieve Na+-free and Cl−-free conditions, NaCl was replaced by LiCl or sodium gluconate (NaGlu). In experiments performed to determine the Na+ dependence of transport, sodium chloride was replaced by an equimolar concentration of choline chloride. To determine whether l-[14C]alanine transport is an electrogenic process, cells were depolarized by the addition of 50 mM KCl or NH4Cl (5); in these experiments, 100 mM sucrose added to Hanks' balancing for the increased osmolarity represented the control situation. 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. Subsequently, cells were solubilized by 0.1% vol/vol Triton X-100 (dissolved in 5 mM Tris·HCl, pH 7.4), and radioactivity was measured by liquid scintillation counting.
Cell monolayers and renal cortical membranes from the WKY and SHR were washed with PBS and 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), with bovine serum albumin 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 60 μg of protein were separated by SDS-PAGE with 10% polyacrylamide gel and then electroblotted onto nitrocellulose membranes (Bio-Rad). Blots were blocked for 1 h with 5% nonfat dry milk in PBS (10 mmol/l PBS) at room temperature with constant shaking. Blots were then incubated with anti-ASCT2 polyclonal antibody (1:800; Chemicon International) in 5% nonfat dry milk in PBS-T (0.01% Tween 20-PBS) overnight at 4°C. The immunoblots were subsequently washed and incubated with fluorescently labeled goat anti-rabbit (1:10,000; IRDye 800, Rockland) or the fluorescently labeled goat anti-mouse secondary antibody (1:5,000; AlexaFluor 680, Molecular Probes) for 60 min at room temperature and protected from light. The membrane was washed and imaged by scanning at both 700 and 800 nm with an Odyssey Infrared Imaging System (LI-COR Biosciences).
One microgram of total RNA was reverse transcribed to cDNA with SuperScript First Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The reverse transcription was performed at 50°C and with the use of 5 μg/μl random hexamers. The ASCT2 cDNA was amplified by PCR using the following set of rat-specific primers: forward 5′-GCC TGA TCG GAG GTG CAG CC-3′ and reverse 5′-CGG GTA AAG AGG AAG TAG ATG-3′, corresponding to nucleotides 334 and 983 of the rat cDNA (GenBank accession no. AJ132846). The B0,+ cDNA was amplified by PCR using the following set of primers: forward 5′-AAC AGT ATT GGG ATA AAG TGA-3′ and reverse 5′-TAA TGG CAT CAG AGT AAC AG-3′, corresponding to nucleotides 755 and 1136 of the rat B0,+ mRNA sequence (GenBank accession no. NM_001037544). PCR was performed with Platinum TaqPCRx DNA Polymerase (Invitrogen). Amplification conditions were as follows: hot start of 2 min at 94°C; 30 cycles of denaturing (94°C for 30s), annealing (55°C for 30 s), and extension (72°C for 45 s); and a final extension of 7 min at 72°C. The PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide.
Real-time PCR quantification of rat ASCT2.
Kidney cortices from WKY and SHR (4 and 12 wk of age) and immortalized renal PTE WKY and SHR cells were homogenized (Diax, Heidolph) in Trizol reagent (75 mg/ml; Invitrogen), and total RNA was extracted according to the manufacturer's instructions. All animal interventions were performed in accordance with the European Directive no. 86/609 and the rules of the Guide for the Care and Use of Laboratory Animals, 7th ed, Washington, DC. Instittue for Laboratory Animal Research (ILAR), 1996. The RNA preparation was further treated with DNase (Ambion), to eliminate potential genomic DNA contamination. Reverse transcription was performed with SuperScript First Strand System for RT-PCR (Invitrogen), using 5 μg/μl random hexamers as primers at 50°C, according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA in a total volume of 20 μl. Standards for ASCT2 and GAPDH were obtained by conventional PCR amplification, using Platinum TaqPCRx DNA Polymerase (Life Technologies) and the following rat-specific primers: rASCT2 forward primer 5′-CGT CCT CAC TCT TGC CAT CAT-3′ and reverse primer 5′-CCA AAA GCA TCA CCC TCC AC-3′ (nucleotide positions 1298 and 1427 in rat ASCT2 sequence NM_175758); rGAPDH forward primer 5′-GGC ATC GTG GAA GGG CTC ATG AC-3′ and reverse primer 5′-ATG CCA GTG AGC TTC CCG TTC AGC-3′ (nucleotide positions 1348 and 1512 in rat GAPDH sequence M17701). PCR products were gel purified with Qiaex II (Qiagen), quantified by spectrophotometry at 260 nm, and further diluted accordingly in serial steps. All PCR fragments were cloned and sequenced. Real-time PCR was carried out using a LightCycler (Roche, Mannheim, Germany). Each RT-PCR reaction mixture (50 μl) included reverse transcription products corresponding to 50 ng of total RNA or standard DNA, 1× SYBR Green I master mix (LightCycler FastStart DNA MasterPLUS SYBR Green I, Roche), and 0.5 μM each forward and reverse primer, mentioned above. Cycling conditions were as follows: denaturation (95°C for 1 min), amplification and quantification (95°C for 10 s, 60°C for ASCT2 and 62°C for GAPDH 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 35 times, a melting curve program (65–95°C with a heating rate of 0.1°C/s and continuous fluorescence measurement), and a cooling step to 40°C. Amplification specificity was checked using melting curves, following the manufacturer's instructions. In addition, PCR products were separated by electrophoresis in a 2% TBE agarose gel to confirm that correct band sizes were obtained. Target mRNAs were quantified by measuring the threshold cycle (when fluorescence is statistically significantly above background) and reading against a calibration curve. Results were analyzed with LightCycler Software v.3.5 (Roche Applied Science, Mannheim, Germany) using the second derivate maximum method. The relative amount of each mRNA was normalized to the housekeeping gene (GAPDH) mRNA. Each sample was tested in duplicate.
l- and d-Amino acids, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), and N-(methylamino)-isobutyric acid were purchased from Sigma Chemical (St. Louis, MO). l-[14C]alanine (specific activity 152 mCi/mmol) was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK).
Km and maximum velocity (Vmax) values for the uptake of l-[14C]alanine were determined from a competitive uptake inhibition protocol (10) and calculated from nonlinear regression analysis using the GraphPad Prism statistics software package (21). For calculation of the IC50, the parameters of the equation for one-site inhibition were fitted to the experimental data (21). Arithmetic means are given with SE. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Newman-Keuls test for multiple comparisons. A P value < 0.05 was assumed to denote a significant difference.
Inward transfer of l-alanine.
To determine the initial rates of uptake, SHR and WKY PTE cells were incubated with a nonsaturating (0.25 μM) concentration of l-[14C]alanine for 1, 3, 6, 12, 30, and 60 min. In both types of cells, uptake of nonsaturating concentration of l-[14C]alanine was linear with time up to 60 min of incubation (Fig. 1). As depicted in Fig. 1, the initial rate for l-[14C]alanine uptake was significantly lower in SHR than in WKY PTE cells.
Since transfer of neutral amino acids across the plasma membrane can be mediated by both Na+-dependent and Na+-independent transport systems, a set of experiments was performed replacing NaCl with an equimolar concentration of choline chloride to determine a potential Na+ dependence of l-[14C]alanine apical uptake. Na+ removal from the uptake solution almost completely abolished transport activity in both SHR and WKY PTE cells (Fig. 2A). Complete Na+ activation curves are shown in Fig. 2B. Linearization of data according to the Hill equation yielded a Hill coefficient of 0.98 and 1.0 for SHR and WKY PTE cells, respectively. A more detailed analysis of the Na+-dependent l-[14C]alanine uptake using Lineweaver-Burk plots revealed the presence of high- and low-affinity uptake processes in both WKY and SHR PTE cells (Fig. 3, A and B). The kinetic parameters (Km and Vmax) for the high- and low-affinity Na+-dependent l-[14C]alanine uptake in WKY and SHR PTE cells are given in Table 1. The effect of BCH on high- and low-affinity Na+-dependent l-[14C]alanine uptake was also evaluated. In the WKY PTE cells, BCH induced a decrease in the affinity for Na+ without changes in Vmax values, in both low- and high-affinity components, thus behaving as a competitive inhibitor (Table 1). In the SHR PTE cells, the effects of BCH on Km and Vmax for the high- and low-affinity components of Na+-dependent l-[14C]alanine uptake did not attain a statistical significance (Table 1).
The replacement of NaCl by LiCl reduced the transport activity by ∼90% in both SHR and WKY PTE cells (Fig. 4A). Replacing NaCl with NaGlu produced a significant reduction in l-alanine uptake in both WKY and SHR PTE cells, this being more marked in the latter (Fig. 4A). The addition of 50 mM KCl, but not of 50 mM NH4Cl, to the uptake solution, a manipulation that reduces cell membrane potential, resulted in a slight but statistically significant (P < 0.05) reduction (∼13% decrease) in l-[14C]alanine uptake (Fig. 4B), suggesting that alanine uptake occurs mainly through nonelectrogenic transporters.
In the presence of extracellular 140 mM Na+, the system A inhibitor N-(methylamino)-isobutyric acid (MeAIB) had no inhibitory effect on l-alanine accumulation in both types of cells (WKY, 91 ± 8%, and SHR, 115 ± 7% of control).
Subsequent experiments were designed to determine the apparent kinetics of l-alanine transporters under Na+ Vmax experimental conditions (extracellular 140 mM Na+). Cells were incubated for 6 min in the absence or presence of increasing concentrations of unlabeled substrate (3–3,000 μM). The effect of BCH on the kinetic parameters was also investigated. In both types of cells, the accumulation of l-[14C]alanine was found to be concentration dependent and a saturable process (Fig. 5). The apparent kinetic parameters of l-[14C]alanine uptake, determined by nonlinear analyses of the inhibition curves, are given in Table 2. The transport capacity of the saturable component of l-[14C]alanine transport in SHR was lower than that for WKY PTE cells, at all substrate concentrations with similar Km values (Table 2). Experiments were also conducted in the presence of BCH to reduce the contribution of l-type amino acid transport. In the presence of BCH, Km values for l-[14C]alanine uptake in SHR PTE cells were markedly increased without changes in Vmax, whereas Km values in WKY PTE cells changed only slightly (Table 2).
Substrate selectivity of l-alanine uptake was investigated by inhibition experiments in which the accumulation of 0.25 μM l-[14C]alanine was measured in the presence of 1 mM unlabeled amino acids and selective analogs (Fig. 6A). In both cell lines, l-alanine uptake was markedly (>80%) inhibited by l-isomers of neutral amino acids, such as alanine, serine, threonine, and cysteine, which is consistent with amino acid transport through ASCT2. Nevertheless, the profile of inhibition differs considerably in the case of other neutral and aromatic amino acids, such as leucine, isoleucine, phenylalanine, methionine, tyrosine, and histidine, which, in WKY PTE cells, produced moderate inhibition (10–50%). By contrast, in SHR PTE cells, neutral and aromatic amino acids produced a high degree of inhibition (75–90%) on l-[14C]alanine uptake. The basic amino acids lysine and arginine also reduced the accumulation of l-[14C]alanine in SHR PTE cells. In WKY PTE cells, l-alanine uptake was also inhibited by glutamine and asparagine.
To prove that the uptake of l-[14C]alanine occurs through system ASC, substrate specificity was examined in the presence of BCH 3 mM. As shown in Fig. 6B, the SHR cell profile of inhibition changed considerably, becoming less sensitive to glycine, isoleucine, phenylalanine, methionine, tyrosine, and histidine (0–35% inhibition) as well as to cationic amino acids. By contrast, glutamine was able to reduce significantly l-[14C]alanine uptake in SHR PTE cells. WKY PTE cells were less affected by the presence of 3 mM BCH in the uptake solution.
These results indicate that l-alanine transport in SHR and WKY PTE cells is largely promoted through a high-affinity, Na+-dependent, Li+-sensitive amino acid transporter, insensitive to amino acid analog MeAIB, with specificity for alanine, serine, and cysteine. These are features of ASC-like activity. As evidenced by Vmax values, the Na+-dependent and Li+-sensitive transport of l-[14C]alanine was lower in SHR than in WKY PTE cells. However, in SHR PTE cells, l-alanine transport was sensitive to BCH as well as to basic amino acids, and the absence of Cl− in the media inhibited the uptake. These observations suggest that, in SHR PTE cells, the Na+-dependent component might be the combination of system B0,+ and ASCT2.
Expression of ASCT2.
The presence of ASCT2 protein in SHR and WKY PTE cells was studied by means of immunoblotting using an antibody raised against ASCT2. As shown in Fig. 7, antibodies against ASCT2 recognized the presence of the protein in immortalized WKY and SHR PTE cells and in renal cortical membranes from WKY and SHR. The abundance of ASCT2 (corrected for β-actin) was lower in SHR than in WKY PTE cells (Fig. 7A). The reduced ASCT2 protein expression in SHR PTE cells correlates positively with the lower transport capacity observed in SHR PTE cells compared with WKY PTE cells. The reduced ASCT2 protein expression in the SHR was also observed in renal cortical membranes in both 4- and 12-wk-old rats (Fig. 7, B and C).
ASCT2 transcript abundance.
Detection of ASCT2 transcript in immortalized PTE cells and kidney cortices from WKY and SHR at 12 wk of age was performed by conventional RT-PCR, using rat-specific primers. As depicted in Fig. 8A, all samples amplified the expected 650-bp fragment. Transcript abundance of ASCT2 was measured by quantitative real-time PCR in immortalized PTE cells and kidney cortices from WKY and SHR. The expression of the ASCT2 transcript was normalized to that of the housekeeping gene GAPDH, which was identical in WKY and SHR. Data are presented as the ratio of ASCT2 to GAPDH. The mRNA expression of ASCT2 was lower (P < 0.05) in WKY than in SHR PTE cells (Fig. 8B), which is not consistent with higher ASCT2 protein in WKY than in SHR PTE cells. Posttranscriptional events may be responsible for the low expression of ASCT2 protein in SHR cells, as previously reported by Tailor et al. (36). As depicted in Fig. 8C, the expression of ASCT2 transcript was markedly lower in SHR than in WKY rat kidney cortices, at both 4 and 12 wk of age, which correlated well with differences in ASCT2 protein expression between WKY and SHR. ASCT2 mRNA decreased with age in both WKY and SHR.
Detection of B0,+ transcript.
To explore the possible involvement of system B0,+, a Na+- and Cl−-dependent amino acid transporter sensitive to BCH as well as neutral and basic amino acids (35, 41), conventional RT-PCR was performed in immortalized PTE cells and kidney cortices from WKY and SHR. A specific primer set, designed based on the rat B0,+ sequence NM_00103744, was used. As shown in Fig. 9, the expected 400-bp fragment corresponding to B0,+ was present only in the SHR immortalized PTE cells.
The present study shows that renal WKY and SHR PTE cells take up l-[14C]alanine mainly through the high-affinity Na+-dependent amino acid transporter system ASCT2. The SHR PTE cells were found to be endowed with lower expression level and function of ASCT2. Furthermore, findings described here in immortalized WKY and SHR PTE cells are consistent with that occurring in vivo in WKY and SHR. In fact, the abundance of ASCT2 transcript and protein in kidney cortices was also markedly lower in SHR than in normotensive WKY.
Although the expression of ASCT2 in renal epithelial cells has been reported previously (3, 11, 15), it was only recently that functional evidence for ASC-like activity in the apical membrane of kidney epithelial cells was observed. Oppedisano and co-workers observed transport activity with characteristics of ASCT2 (22, 23) in liposomes obtained from rat renal apical plasma membranes. The immortalized renal PTE cells from WKY and SHR are well-established models in our laboratories (25, 26, 29, 30) that have been used to evaluate both the diversity and the regulation of amino acid transport systems (29). In the present study, l-alanine was used to assess the presence of ASCT2, one of the transport systems responsible for the Na+-dependent inward transfer of this amino acid. However, transport systems B0 and ASCT2 are most likely to be responsible for the Na+-dependent uptake of alanine in kidney brush border membranes. WKY and SHR PTE cells transport quite efficiently l-alanine through the apical cell border, and several findings suggest that this uptake process is a facilitated mechanism and proceeds through ASCT2. Most of l-[14C]alanine entered the cells in a Na+-dependent manner, and only a minor component of l-[14C]alanine uptake (∼10–15%) was found not to require extracellular Na+. The Na+ activation Hill coefficient of unity indicates a 1:1 Na+-to-alanine activation stoichiometry for secondary active transport in both cell lines. However, in-depth analysis of this process revealed the presence of high- and low-affinity states for the Na+-dependent l-[14C]alanine uptake in both cell lines. Although this could be interpreted as the presence of two transporter entities, it is likely that this is not the case. In fact, at low extracellular Na+ concentrations, the Na+-dependent l-[14C] alanine uptake in both WKY and SHR PTE cells is a high-affinity, low-capacity process, and increases in extracellular Na+ reduced the affinity for the substrate but increased the capacity to take up l-[14C]alanine. Another finding that supports this suggestion is that BCH decreases the affinity of the transporter but does not affect the Na+-dependent l-[14C]alanine uptake, this being particularly evident for WKY PTE cells. It is also likely that differences in the Na+-dependent l-[14C]alanine uptake between WKY and SHR cells may be related to the presence of two different Na+-dependent l-[14C]alanine transporters in the latter, as discussed below.
The l-[14C]alanine uptake was unaffected by MeAIB, suggesting that inward transfer in WKY and SHR PTE cells was not promoted by the system A. BCH only reduced l-[14C] alanine uptake ∼25%; this low level of sensitivity to BCH supports the view that l-alanine transport is mediated by an ASC-like transporter. Furthermore, l-[14C]alanine uptake was also found to be markedly inhibited by Li+. Small neutral amino acids such as alanine, serine, and cysteine significantly inhibited the uptake of l-[14C]alanine in both cell lines. System B0 is largely electrogenic, with high affinity for phenylalanine (7). The uptake of l-[14C]alanine in WKY and SHR PTE cells was largely nonelectrogenic. In the presence of BCH (to exclude the role of system L), l-[14C]alanine uptake in SHR and WKY PTE cells was less sensitive to phenylalanine than to alanine. Taken together, these results indicate that the Na+-dependent alanine uptake in SHR and WKY cells proceeds mainly through the Na+-dependent neutral amino acid ASCT2 transporter and not by systems B0 and A. This suggestion agrees with data in previous reports that characterized the Na+-dependent alanine uptake in renal epithelial cells (19, 34).
In SHR PTE cells, the ability of the transporter to take up l-[14C]alanine appeared to be lower than that observed in WKY PTE cells. As shown in time course experiments, the initial rate of l-[14C]alanine uptake in SHR PTE cells was already two times lower than that in WKY PTE cells. In addition, the saturable component of Na+-dependent l-[14C]alanine transport under Vmax conditions in SHR was one-half that in WKY PTE cells. In the presence of BCH, the system L inhibitor, Km values for transport increased and became similar in both cell lines. The Km values reported here are in close agreement with those described previously for human ASCT2 expressed in Xenopusoocytes (∼169.7 μM) (24) and for the pig kidney epithelial cell line LLC-PK1 (380 μM) (19). Furthermore, differences in the magnitude of Na+-dependent l-[14C]alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in the expression of ASCT2 protein, this being more abundant in WKY than in SHR PTE cells. The discrepancy between mRNA concentration and protein expression observed in SHR PTE cells might be related to posttranscriptional events. Several levels of nuclear posttranscriptional events can be regulated, such as the control of splicing efficiency, precursor RNA stability, polyadenylation, or RNA transport (2). Whether this overproduction in SHR involves a cis- or trans-regulatory mechanism or whether any labile protein factor affected the regulation is unknown. Studies to elucidate the molecular mechanism of mASCT2 overproduction in SHR PTE cells are required.
Different routes for alanine uptake are present in SHR PTE cells. SHR cells, but not WKY cells, were also found to take up l-[14C]alanine in a Cl−-dependent manner (∼45% of l-alanine uptake) that, in the absence of BCH, was sensitive to inhibition by leucine, isoleucine, phenylalanine, methionine, tyrosine, and histidine and to the cationic amino acids lysine and arginine. The Na+- and Cl−-dependent l-[14C]alanine transporter most likely involved corresponds to system B0,+, a transporter sensitive to BCH, neutral and basic amino acids (35, 41). Thus the major Na+-dependent l-alanine transporter in WKY cells is ASCT2, contributing to ∼85% of the total l-alanine uptake. By contrast, in SHR cells Na+-dependent component may result of ASCT2 (∼55%) and system B0,+ (∼45%). A minor contribution to l-alanine uptake by Na+-independent transporters is also observed in both cell lines.
To determine whether the findings obtained in immortalized WKY and SHR PTE cells might reflect the in vivo situation, a set of experiments was conducted in renal cortices from SHR and WKY rats of 4 and 12 wk of age. An ASCT2-specific fragment was detected in the mRNA samples from the rat kidney, suggesting that this transporter was also expressed in renal cortices. The quantitative real-time PCR experiments correlated positively with data from immunoblots, which indicated that the expression of ASCT2 in renal cortices from SHR was lower than that in the WKY. This suggests that immortalized SHR and WKY PTE cells constitute a good experimental model for the study of ASCT2.
Neutral amino acid transporter ASCT2 displays substrate-induced Na+ antiport activity; therefore, as an obligatory exchanger, it cannot mediate net amino acid uptake (6). Thus the role of ASCT2 in proximal tubule homeostasis is that of a mechanism for the delivery of glutamine for ammoniagenesis (4, 8) and for the removal of other small neutral amino acids from the extracellular space, maintaining their low extracellular levels. ASCT2 belongs to a restricted group of transporters that share specificity for glutamine, since glutamine is the major precursor of urinary ammonia, thus playing a key role in acid-base homeostasis. Interestingly, however, glutamine was higher in muscle and plasma of SHR at 6 wk of age and thereafter (12). These differences, because they occurred most strikingly in SHR during the prehypertensive state, were suggested to be related to the development of hypertension (12). However, ammonium urinary excretion was identical in WKY and SHR (31). The possibility that the SHR uses less glutamine in renal ammoniagenesis because of underexpression of ASCT2 needs to be evaluated.
Another interesting observation is that ASCT2 has been shown to be regulated by nitric oxide (NO) in the human intestinal cell line Caco-2 (39). NO is inactivated by reaction with superoxide (O2−) to produce peroxynitrite. In the kidney of SHR the regulation of renal oxygen consumption by NO is impaired (1), due to the increased superoxide production observed in this model of hypertension (33, 42, 43). NO availability in the kidney is decreased in SHR, resulting in increased oxygen consumption. By lowering intrarenal oxygen levels, reduced NO may contribute to susceptibility to renal injury (1). Taken together, these observations and those described in the present study suggest that, in SHR, oxidative stress might be downregulating ASCT2 by decreasing intrarenal NO availability. Therefore, the modulation of renal ASCT2 transporter in hypertension is worthy of further attention.
In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na+-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells. As a compensatory mechanism, in SHR cells, l-alanine is also transported by other amino acid transport systems, namely B0,+, that account for ∼45% of total Na+-dependent l-[14C]alanine uptake. Finally, findings obtained in immortalized cells match those in vivo: ASCT2 is underexpressed at the kidney cortex level in the SHR.
This work was supported by Fundação para a Ciência e a Tecnologia, POCTI, POCI, FEDER and Programa Comunitário de Apoio (POCI/SAU-OBS/57916/2004).
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