点击显示 收起
【摘要】 The intrarenal autocrine-paracrine dopamine (DA) system is critical for Na + homeostasis. L -Dihydroxyphenylalanine ( L -DOPA) uptake from the glomerular filtrate and plasma provides the substrate for DA generation by the renal proximal tubule. The transporter(s) responsible for proximal tubule L -DOPA uptake has not been characterized. Renal cortical poly-A + RNA injected into Xenopus laevis oocytes induced L -DOPA uptake in a time- and dose-dependent fashion with biphasic K m s in the millimolar and micromolar range and independent of inward Na +, K +, or H + gradients, suggesting the presence of low- and high-affinity L -DOPA carriers. Complementary RNA from two amino acid transporters yielded L -DOPA uptake significantly above water-injected controls the rBAT/b 0,+ AT dimer (rBAT) and the LAT2/4F2 dimer (LAT2). In contradistinction to renal cortical poly-A +, L -DOPA kinetics of rBAT and LAT2 showed classic Michaelis-Menton kinetics with K m s in the micromolar and millimolar range, respectively. Sequence-specific antisense oligonucleotides to rBAT or LAT2 (AS) caused inhibition of rBAT and LAT2 cRNA-induced L -DOPA transport and cortical poly-A + -induced arginine and phenylalanine transport. However, the same ASs only partially blocked poly-A + -induced L -DOPA transport. In cultured kidney cells, silencing inhibitory RNA (siRNA) to rBAT significantly inhibited L -DOPA uptake. We conclude that rBAT and LAT2 can mediate apical and basolateral L -DOPA uptake into the proximal tubule, respectively. Additional L -DOPA transport mechanisms exist in the renal cortex that remain to be identified.
【关键词】 sodium balance amino acid transport proximal tubule
THE INTRARENAL AUTOCRINE - PARACRINE dopamine (DA) system is an important mediator of natriuresis in mammalian Na + homeostasis. There is strong evidence that urinary DA is primarily generated in the kidney and not derived from the plasma or from dopaminergic neurons innervating the renal parenchyma ( 31 ). Because the kidney lacks the ability to hydroxylate tyrosine, the starting substrate in the renal DA synthetic pathway is L -dihydroxyphenylalanine ( L -DOPA). L -DOPA is taken up by the renal proximal tubule ( 32, 38 ) and decarboxylated into DA by aromatic amino acid decarboxylase ( 34 ). The proximal tubule (PT) seems to possess most of the decarboxylase activity and is the principal site of DA synthesis in the kidney ( 13, 14 ). In contrast to the origin of renal DA synthesis, the site of DA action in the kidney is far from unique. All five isoforms of the DA receptors are found in the kidney and are distributed extensively in the vasculature and the renal epithelia ( 5 ). In the kidney, DA can regulate renal plasma flow ( 20 ), glomerular filtration ( 20 ), tubuloglomerular feedback ( 14 ), renin release ( 1, 2 ), and epithelial ion and water transport ( 22, 35 ).
Considering the plethora of actions of DA in the kidney, the understanding of its synthesis assumes paramount importance. DA synthesis in the PT is regulated by acute as well as chronic changes in extracellular fluid volume induced by saline infusion ( 18 ) and increased dietary salt intake ( 8, 21 ), respectively. The mechanism by which the intrarenal DA system senses and reacts to changes in effective extracellular fluid volume is poorly understood. The current data indicate that there is interplay between the DA system and other intrarenal paracrine networks such as the local renin-angiotensin ( 2 ) and adenosine systems ( 36 ). In addition, the decarboxylation of L -DOPA to DA appears to be modulated by changes in dietary salt ( 27, 30 ). To fully understand the regulation of DA genesis by the PT, one needs to commence at the very first step, the uptake of L -DOPA into the PT.
Baines and Chan ( 3 ) showed that when L -[ 3 H]DOPA was infused either from the peritubular or luminal side of the proximal tubule, a significant percentage of the injected L -[ 3 H]DOPA was converted into DA and excreted in the urine ( 3 ). The process was intact in the denervated kidney, suggesting that this was not a neurogenic mechanism. L -DOPA uptake by the proximal tubule has been characterized in one study using micropuncture by Chan showing L -DOPA uptake by the PT cell to be carrier mediated, stereospecific, energy dependent, pH independent, and competitively inhibited by L -phenylalanine ( 4 ). There are also studies addressing L -DOPA uptake using renal cortical slices ( 32 ) as well as cultured renal cell lines ( 9, 34 ). From this body of literature, one or more models have been constructed depicting a carrier-mediated process of L -DOPA uptake in both the apical and basolateral membranes. However, the actual identities of the L -DOPA transporters remain elusive. In recent years, a large number of complementary DNAs from the proximal tubule have been identified to mediate various amino acid transport processes when expressed in heterologous systems ( 37 ). In this study, we examined several candidate amino acid transporters for their ability to take L -DOPA as a substrate in Xenopus laevis oocytes and compared their characteristics to poly-A + RNA from the renal cortex. We conclude that L -DOPA uptake into the PT is mediated by apical rBAT and basolateral LAT2. Other yet unidentified transporters also participate in L -DOPA uptake.
EXPERIMENTAL PROCEDURES
RNA preparation. Native RNA from rat renal cortex was isolated by homogenizing dissected renal cortex in GTC [in M: 4 guanidium thiocyanate, 0.1 2-mercaptoethanol, 0.025 Na 3 citrate (pH 7.0), wt/vol 0.5% N -lauroyl-sarcosine, and RNA was phenol/chloroform-extracted and ethanol-precipitated. Poly-A + RNA was isolated by oligo-dT affinity chromatography and size-fractionated by a continuous sucrose density gradient (6-20% wt/vol). The range of kilobases recovered from each fraction was confirmed by formaldehyde gel electrophoresis with RNA ladder. Complementary RNAs (5'-methyl-capped) were transcribed in vitro from linearized cDNA by in vitro transcription kit. Plasmids containing the sequence for rBAT and LAT2, respectively, were linearized by restriction enzyme cut and cRNA synthesized using T7 and SP6 RNA polymerases, respectively. Integrity and size of cRNAs were confirmed by ethidium bromide gel electrophoresis. Complementary DNAs were kindly supplied by Dr. F. Verrey (University of Zürich, Zürich, Switzerland): 4F2hc and rBAT in pSPORT1, hLAT1 and hLAT2in pSDEasy.
Expression and uptake in X. laevis oocytes. Oocytes were manually dissected from X. laevis (Nasco, Fort Atkinson, WI), and ovarian tissues were defollicularized with collagenase D (2 mg/ml; Boehringer Mannheim, Indianapolis, IN) in OR II medium (in mM: 82.5 NaCl, 2 KCl, 1 MgCl 2, 10 HEPES/Tris, pH 7.5) and stored in modified Barth's solution [in mM: 88 NaCl, 1 KCl, 0.82 MgSO 4, 0.41 CaCl 2, 0.33 Ca(NO 3 ) 2, 2.4 NaHCO 3, 10 HEPES/Tris, pH 7.40, 20 mg/ml gentamicin] at 18°C. Oocytes were injected (Drummond microinjector, Broomall, PA) with 67 nl of water with or without 5-10 ng of cRNA or 30 ng of poly-A + RNA. Injected oocytes were incubated in Barth's solution for 3-4 days at 18°C before flux measurements. Before the experiment, oocytes were washed in rinse solution (in mM: 88 choline Cl, 2 KCl, 1 CaCl 2, 1 MgCl 2, 10 HEPES/Tris, pH 7.5). For timed isotope uptake, oocytes were incubated in uptake solution (in mM: 88 NaCl, 2 CaCl 2, 2 MgCl 2, 20 HEPES/Tris, pH 7.4, 3.5 µCi 0.01-2 L -[ 3 H]DOPA) for the specified time at 20°C. Isotope uptake was stopped by washing the oocytes with the rinse solution supplemented with 5 mM L -DOPA at 4°C. Individual oocytes were dissolved in 10% SDS and radioactivity was determined by scintillation counting. For dose-related isotope uptake, oocytes were injected with either 5, 10, 20, and 40 ng of renal cortical poly-A + 3 days before the uptake assay. For cation dependence, choline was substituted isoosmotically in the uptake solution by Na + or K +, and pH was adjusted to either 7.4 or 5.4. In experiments examining trans -activation of LAT2 by phenylalanine, oocytes were injected with 1 nmol of L -phenylalanine 6 h before isotope uptake. For kinetic experiments, separate groups of oocytes were incubated in uptake solution containing varying concentrations of unlabeled L -DOPA (0.01, 0.05, 0.100, 0.200, 0.250, 0.500, 1.00, and 2.0 mM) for 15 min for flux calculation. Michaelis-Menton kinetics were analyzed by Eadie-Hofstee transformation (Sigmaplot, Rockware, Golden, CO) to approximate K m and V max. Antisense oligonucleotides were synthesized to be complementary to the first 21-29 coding nucleotides of rBAT and LAT2 and annealed to the RNA (55°C x 10 min and then 4°C x 20 min) to block translation (5'-GTCCTCATTCATGTCTATCCG-3' for rat rBAT, 5'-GGTTCCCTTTTCCATCTTTCTCCGTAGGG-3' for rat LAT2). Separate groups of oocytes were injected with either renal cortical poly-A +, rBAT cRNA, and LAT2 cRNA with or without 15 x molar excess of AS for rBAT or AS for LAT2 followed by isotope uptake determination.
Silencing inhibitory RNA experiment. Silencing inhibitory RNA target sites were chosen by scanning the OKP rBAT mRNA sequence for AA dinucleotides, recording the 19 nucleotides immediately downstream of the AA, and then comparing the potential siRNA target sequences with an appropriate genome database to eliminate any sequences with significant homology to other genes. After selecting the OKP rBAT siRNA target site, a DNA oligonucleotide consisting of a 19-nucleotide sense siRNA sequence linked to its antisense complementary siRNA sequence by a short spacer (TTCAAGAGA) was designed. Nucleotide overhangs to the Bam HI and Hind III restriction sites were added to the 5'- and 3'-end of the oligonucleotides to be cloned into pSilencer 2.0 or 3.0. Two different siRNAs were designed and named siRNA 20 and siRNA 54. After being cloned, the plasmid containing the oligonucleotide sequence for OKP rBAT siRNA was transfected into OKP cells in culture following the Lipofectamine Plus procedure from Invitrogen using cells plated in six-well plates. Seventy to eighty percent of tranfection efficiency was documented in GFP gene containing mammalian cell expression vector-transfected OKP cells by immunofluorescence microscopy quantification before L -DOPA uptake studies were performed.
L -DOPA uptake in mammalian culture cells. L -DOPA uptake was initiated by the addition of 1 ml of Hanks' medium (137 mM NaCl, 5 mM KCl, 0.8 mM MgSO 4, 0.33 mM Na 2 HPO 4, 0.44 mM KH 2 PO 4, 0.25 mM CaCl 2, 1.0 mM MgCl 2, 0.15 mM Tris·HCl, 1.0 mM sodium butyrate, pH 7.4) with 4.7 µCi of 3 H- L -DOPA and 100 µM unlabeled L -DOPA. Cells were incubated at room temperature for 15 min. Uptake was terminated by rapid removal of uptake solution by means of vacuum pump connected to a Pasteur pipette followed by two washes with ice-cold Hanks' medium containing 5 mM unlabeled L -DOPA. Five-hundred microliters of 4% SDS were added to each well of cells for lysis. Twenty microliters of aliquot were used for protein concentration determination. The rest was used for determination of L -[ 3 H]DOPA in scintillation counter. Final uptake for each sample was expressed in counts per minute per milligram of protein per 15 minutes.
RESULTS
Rat renal cortical poly-A + RNA induces L -DOPA uptake in X. laevis oocytes. Renal cortical poly-A + RNA induced time- and concentration-dependent L -DOPA uptake in oocytes ( Fig. 1, A and B, respectively). Oocytes injected with water and no poly-A + RNA were associated with L -DOPA transport that was always substracted in later L -DOPA uptake experiments. L -DOPA transport was independent of Na +, K +, or pH gradients (data not shown). This indicated that there are RNA sequences in the renal cortex coding for an L -DOPA carrier. Injection of size-fractionated renal cortical poly-A + RNA showed a single peak residing in the fractions corresponding to the transcript in the 2- to 4-kb range ( Fig. 2 ), which encompasses most of the amino acid transporters. Analysis of substrate kinetics showed a complex pattern ( Fig. 3 A ), and a linear transformation of the data ( Fig. 3 B ) yielded two K m s corresponding to a high-affinity (10-25 µM) and a low-affinity ( 1 mM) component, suggesting that there is more than one L -DOPA transporter in the renal cortex. Given the structural similarity between L -DOPA and phenylalanine and previous data showing competitive inhibition of the L -DOPA transport in the proximal tubule by L -phenylalanine ( 4 ), we screened three cloned amino acid transporters as putative candidates for L -DOPA transporters.
Fig. 1. Poly-A + RNA-induced L -dihydroxyphenylalanine ( L -DOPA) uptake. Xenopus laevis oocytes were injected with 20 ng of poly-A + RNA from rat kidney cortex, and L -[ 3 H]DOPA uptake was measured by isoptope flux 3-4 days later. Two typical experiments are shown with each data point representing the means ± SD of 8-12 oocytes. A : time course of uptake. B : dose dependence. Three independent sets of experiments were perfomed with similar results.
Fig. 2. L -DOPA uptake induced by different size fractions of renal cortical poly-A +. Poly-A + RNA from rat kidney cortex was size-fractionated by sucrose density gradient and injected into X. laevis oocytes, and L -[ 3 H]DOPA uptake was measured by isoptope flux 3-4 days later. Bottom : ethidium bromide-stained RNA gel of the fraction corresponding to each injection. Each data point represents the means ± SD of 8-12 oocytes.
Fig. 3. Kinetics of L -DOPA uptake induced by renal cortical poly-A + RNA. X. laevis oocytes were injected with poly-A + RNA from rat kidney cortex, and L -[ 3 H]DOPA uptake was measured by isoptope flux in the presence of varying concentrations of L -DOPA (0.01, 0.05, 0.1, 0.2. 0.25, 0.5, 1, and 2 mM). A : one typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Background L -DOPA transport in the water-injected oocytes group was substracted from the poly-A + RNA-injected oocytes group. Three independent studies were performed with similar results. B : Eadie-Hofstee transformation of the data that yielded 2 K m s: a high-affinity (10-25 µM) and a low-affinity ( 1 mM) in 3 independent experiments.
Oocytes injected with rBAT cRNA showed sevenfold higher L -DOPA uptake than the water-injected controls ( see Fig. 5 ). Coinjection of rBAT and the subunit b 0,+ AT further induces L -DOPA uptake to about ninefold of that of water. Arginine uptake was used as a positve control for rBAT ( Fig. 4 ). This is not an unexpected finding as oocytes express an endogenous homolog of b 0,+ AT ( 11 ). Similar to cortical poly-A + RNA, the rBAT-induced L -DOPA uptake is also independent of inwardly directed Na +, K +, and pH gradients ( Fig. 5 ). This suggests that rBAT induces expression of an L -DOPA carrier with functional similarities to the one in renal cortical poly-A + RNA.
Fig. 4. L -DOPA uptake induced by renal cortical poly-A + RNA, rBAT, and rBAT/b 0,+ AT. X. laevis oocytes were injected with poly-A + RNA from rat kidney cortex or cRNA, and 3 H- L -DOPA uptake was measured by isoptope flux 3-4 days later ( left ). One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results. Positive control was done with L -arginine uptake ( left ).
Fig. 5. Cation and H + gradient dependence of L -DOPA uptake induced by rBAT cRNA. X. laevis oocytes were injected with renal cortical poly-A + RNA or rBAT cRNA, and 3 H- L -DOPA uptake was measured by isoptope flux. One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. The cationic concentration (mM) and pH of the uptake solution are shown. Three independent experiments were performed with similar results.
To further examine whether the rBAT sequence accounts for the L -DOPA uptake induced by renal cortical poly-A + RNA, we compared the substrate kinetics of rBAT-induced L -DOPA uptake to that of renal cortical poly-A + RNA-induced L -DOPA uptake ( Figs. 3 and 6 ). Although renal cortical poly-A + RNA-induced uptake can be resolved into two components with two K m s (10-25 µM and 1 mM), rBAT cRNA shows classic Michaelis-Menton kinetics with a single K m of 25 µM ( Fig. 6 ). It is possible that the high-affinity L -DOPA transporter in renal cortical poly-A + RNA is rBAT. We used a second approach to delineate this fact. We reduced rBAT contribution to poly-A + -induced L -DOPA transport by blocking rBAT translation with rBAT-specific antisense oligonucleotides. Antisense oligonucleotides in proximity of the start codon were annealed with rBAT cRNA or rat renal cortical poly-A + RNA, injected into oocytes, and the effect on L -DOPA uptake was assessed ( Fig. 7 ). Antisense oligonucleotide rBAT blocks 30% of the L -DOPA uptake induced by poly-A + RNA and 80% of the L -DOPA or L -arginine transport induced by rBAT cRNA. Sense or scrambled oligonucleotides had no effect on L -DOPA transport (not shown).
Fig. 6. Kinetics of L -DOPA uptake induced by rBAT cRNA. X. laevis oocytes were injected with rBAT/b 0,+ AT, and L -[ 3 H]DOPA uptake was measured by isoptope flux in the presence of varying concentrations of L -DOPA (0.01, 0.025, 0.050, 0.075, 0.100, 0.5, and 1 mM). One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results. Inset : Eadie-Hofstee transformation of the data that yielded a K m of 25 mM.
Fig. 7. Inhibition of L -DOPA transport by antisense-oligonucleotides specific to rBAT. Renal cortical poly-A + RNA or rBAT cRNA was annealed to excess rBAT-specific antisense oligonucleotides and injected into oocytes. 3 H- L -DOPA uptake was measured by isoptope flux. Right : control using L -arginine uptake. Fifty micromolar unlabeled L -DOPA and L -arginine were used in this experiment, respectively. One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results and statistical significance. *Statistical significance ( P < 0.05 ANOVA).
Oocytes injected with LAT2/4F2 cRNA showed a twofold higher L -DOPA uptake than water-injected oocytes ( Fig. 8 ). In this case, both subunits were coinjected for all the experiments because none of the subunits are expressed in the oocytes. Because LAT2/4F2 can sustain amino acid countertransport, we examined whether trans phenylalanine can stimulate L -DOPA uptake. Injecting oocytes with 1 nmol L -phenylalanine 6 h before uptake studies was not associated with an increase in L -DOPA transport induced by either renal cortical poly-A + or LAT2/4F2 cRNA ( Fig. 9 ). L -DOPA uptake by LAT2/4F2 showed simple Michaelis-Menton kinetics with a K m of 6 mM ( Fig. 10 ). Coinjection of antisense oligonucleotides for LAT2 blocked 30% of poly-A + -induced L -DOPA uptake and 60% of the LAT2 cRNA-induced L -DOPA uptake ( Fig. 11 ). This suggests that LAT2 also accounts for some of the L -DOPA transport induced by renal cortical poly-A +. However, there are other low-affinity L -DOPA carriers to be identified.
Fig. 8. L -DOPA uptake induced by LAT2/4F2 cRNA. X. laevis oocytes were injected with LAT2/4F2 cRNA, and L -[ 3 H]DOPA uptake was measured by isoptope flux. Right : phenylalanine uptake was used as a positive control. One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results.
Fig. 9. Examination for countertransport activity of L -DOPA uptake induced by renal cortical poly-A + RNA or LAT2/4F2. X. laevis oocytes were injected with LAT2/4F2 cRNA, and 3 H- L -DOPA uptake was measured by isoptope flux. One nanomole of phenylalanine or water was injected 6 h before flux measurements. One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results.
Fig. 10. Kinetics of L -DOPA uptake induced by LAT2/4F2. X. laevis oocytes were injected with poly-A + RNA from rat kidney cortex, and L -[ 3 H]DOPA uptake was measured by isoptope flux in the presence of varying concentrations of L -DOPA (0.01, 0.05, 0.1, 0.2, 0.25, 0.5, 1, and 2 mM). One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results. Inset : Eadie-Hofstee transformation of the data that yielded a K m of 6 mM.
Fig. 11. Inhibition of L -DOPA transport by antisense-oligonucleotides specific to LAT2. Renal cortical poly-A + RNA or LAT2/4F2 cRNA was annealed to excess LAT2-specific anitsense and injected into oocytes. 3 H- L -DOPA uptake was measured by isoptope flux. One typical experiment is shown with each data point representing the means ± SD of 8-12 oocytes. Three independent studies were performed with similar results. *Statistical significance ( P < 0.05 ANOVA).
Because the proximal tubule-like cell line [opossum kidney (OK) cell] also expresses L -DOPA mechanisms, we examined for the contribution of rBAT sequences to this activity on OK cells. Opossum rBAT-specific siRNA sequences from two seperate regions inhibited L -DOPA uptake by 30-40% compared with OK cells transfected with the vector alone ( Fig. 12, A and B ). Knockdown of LAT2 protein was not attempted because of the unavailability of the opossum LAT2 sequence in the database and the fact that the basolateral membane is inaccessible for monolayers on plastic.
Fig. 12. Inhibition of L -DOPA transport in cultured opossum kidney (OK) cells by rBAT-specific silencing inhibitory (si)RNA. Serum-starved confluent OK monolayers were transfected with either the plasmids pSilencer 2.0/3.0 or plasmids containing inserts generating siRNA specific to opossum rBAT sequence 20 or 54 amino acids downstream from the translational start site. 3 H- L -DOPA uptake was measured by isoptope flux. Bars and error bars represent means ± SD of n = 4. *Statistical significance ( P < 0.05 ANOVA) compared with uptake in cells transfected with the corresponding plasmid without insert.
DISCUSSION
The data support the conclusion that rBAT and LAT2 are L -DOPA transporters. Accounting for incomplete efficiency of antisense oligonucleotide inhibition, rBAT and LAT2 probably sustain up to 60-70% of L -DOPA transport in oocytes injected with rat kidney cortex poly-A + mRNA. LAT2 is an amino acid transporter composed of a heavy subunit 4F2hc and a light subunit LAT2 expressed in the basolateral membrane of the proximal tubule allowing exchange of large neutral amino acids ( 10 ). Our data suggest LAT2 is a low-affinity L -DOPA transporter and can possibly account for the basolateral L -DOPA uptake in the proximal tubule cell. rBAT is an amino acid transporter expressed in the apical membrane of the proximal tubule cell and other epithelia composed of a light subunit b 0,+ AT and a heavy subunit rBAT involved in the reabsorption of dibasic amino acids like cystine, arginine, and lysine ( 11 ). rBAT could account for the high-affinity apical L -DOPA uptake by proximal tubule cells.
Proximal tubule L -DOPA uptake has often been described as a Na + -dependent process ( 33 ), but we showed no siginificant Na + dependence in renal cortical poly-A + -, rBAT-, or LAT2-induced L -DOPA transport. The only study in mammalian perfused tubule actually never examined Na + dependence of L -DOPA flux ( 4 ). Most of the older studies assessed L -DOPA uptake by measuring the rate of DA or DOPAC generation or L -DOPA accumulation in kidney cortex slices exposed to varing concentrations of L -DOPA and Na +. The observation of a Na + -induced increase in DA and DOPAC generation can be due to activation of L -DOPA decarboxylation or changes in DA degradation. These studies also showed L -DOPA cellular accumulation even in the absence of Na +, compatible with a Na + -independent mechanism ( 32 ). Renal cortical slices harbor tubular epithelial cells, interstitial and endothelial cells, as well as glomeruli. It is unknown whether cells other than the proximal tubule can take up L -DOPA. Other than direct coupling of L -DOPA transport to Na +, luminal Na + may activate the intrarrenal DA system or stimulate another Na + -coupled apical transport process that secondarily enhances L -DOPA transport.
Recent studies by Soares-da-Silva and co-workers ( 34 ) showed the L -DOPA uptake is Na + independent in LLC-PK 1 cells and mostly Na + independent with a minor Na + -dependent component ( 15%) in OK HC cells ( 10 ). Contrary to renal cortical slices, cell culture models reflect the biology of the tubular epithlial cell without contributions of the other cell types. In addition, inhibition of the amino acid transporter systems L (includes LAT1 and LAT2) was associated with a concentration-dependent decrease in L -DOPA uptake in OK cells and L-type amino acids and dibasic amino acids inhibit L -DOPA uptake in OK cells, suggesting the b 0,+ -amino acid transport system as a L -DOPA carrier ( 10 ). These findings are congruent with our data on LAT2 and rBAT being Na + -independent L -DOPA carriers accounting for more than 50% of L -DOPA uptake in the proximal tubule. Part of the residual renal cortical poly-A + -induced L -DOPA transport in the background of excess antisense oligonucleotides in oocytes may be due to incomplete inhibition of translation as evidenced by residual activity even with rBAT and LAT2 cRNA. However, the magnitude of inihibition by antisense oligonucleotides is less in poly-A +, suggesting the presence of other L -DOPA transporters. Recent published data described TAT1 to be an L -DOPA carrier in the basolateral membrane ( 17 ). A complete list of proximal tubule L -DOPA carriers remains to be assembled.
Patients with inactivating mutations of rBAT ( SLC3A1 gene) have cystine, arginine, and lysine wasting in the urine with the recognizable autosomal recessive clinical phenotype of cystine kidney stones due to the low solubility of cystine ( 7 ). Urinary L -DOPA wasting has never been examined in these patients (6-8, 23). Even though basolateral L -DOPA uptake may compensate and prevent defective DA generation, it is conveivable that L -DOPA wasting may go undetected as do the highly soluble arginine and lysine. An indirect finding in humans suggests that L -DOPA and cystine transport may actually compete with one another in the kidney. Dietary salt has been known to exacerbate cystinuria ( 23 ), and salt restriction has been considered as a therapeutic intervention in patients with this disease ( 15 ). Because dietary Na + loading increases L -DOPA uptake by the proximal tubule, it is conceivable that Na + -loading places increased demand on proximal tubule rBAT to transport L -DOPA, leading to a lower capacity for rBAT to reabsorb cystine. This hypothesis remains to be proved or disproved by subsequent experiments planned for the near future by the author of this paper.
Multiple observations suggest that defects in the intrarenal DA system can contribute to both monogenic and polygenic salt-sensitive hypertension ( 2, 16 ). Defective renal DA generation has been described in humans with low-renin hypertension associated with a volume-expanded state. This is hypothesized to be due to defective renal DA generation as evidenced by lower urinary DA and low urinary DA/ L -DOPA ratios ( 6, 19 ). The DA/ L -DOPA ratio is a surrogate measure of renal DA generation from L -DOPA. Low urinary DA/ L -DOPA suggests a defect may reside either in L -DOPA uptake or conversion from L -DOPA to DA. These patients seem to have exaggerated natriuretic responses to exogenous DA compatible with a chronic compensatory response to suboptimal DA generation ( 26 ). These abnomalitites in urinary DA were not present in patients with high-renin hypertension ( 28 ). Of interest is the fact that normotensive relatives of patients with defective renal DA generation have urinary DA and DA/ L -DOPA ratios that are intermediate between their hypertensive relatives and normal volunteers. These findings suggest that genes encoding proteins responsible for renal L -DOPA uptake and/or DA generation are candidate genes in the current list of contenders accounting for the development of polygenic hypertension. The identification of L -DOPA carriers permits further studies.
GRANTS
This work was supported by National Institutes of Health (NIH) Grants R01-DK-48482 and R01-DK-54392 (to O. W. Moe), the American Heart Association Texas Affiliate (98G-052 to O. W. Moe), and the Department of Veteran Affairs Research Service (O. W. Moe). NIH National Research Service Awards were awared to R. Collazo (T32 DK-07257-17) and to H. Quiñones (T32 DK-07257-2031).
ACKNOWLEDGMENTS
The authors acknowledge the technical expertise of L. A. Crowder.
【参考文献】
Antonipillai I, Broers MI, and Land D. Evidence that specific dopamine-1 receptor activation is involved in dopamine-induced renin release. Hypertension 13: 463-468, 1989.
Asico LD, Ladines C, Fuchs S, Accili D, Carey RM, Semeraro C, Pocchiari F, Felder RA, Eisner GM, and Jose PA. Disruption of the dopamine D3 receptor gene produces renin-dependent hypertension. J Clin Invest 102: 493-498, 1998.
Baines AD and Chan W. Production of urine free dopamine from DOPA: a micropuncture study. Life Sci 26: 253-259, 1980.
Chan YL. Cellular mechanism of renal tubular transport of L -DOPA and its derivatives inn the rat: microperfusion studies. J Pharmacol Exp Ther 199: 17-24, 1976.
Felder CC, McKelvey AM, Gitler MS, Eisner GM, and Jose PA. Dopamine receptor subtypes in renal brush border and basolateral membranes. Kidney Int 36: 183-193, 1989.
Gill JR, Grossman E, and Goldstein DS. High urinary dopa and low urinary dopamine to dopa ratio in salt-sensitive hypertension. Hypertension 18: 614-621, 1991.
Gitomer WL, Reed BY, Ruml LA, Sakhaee K, and Pak CYC. Mutations in the genomic deoxyribonucleic acid for SLC3A1 in patients with cystinuria. J Clin Endocrinol Metab 83: 3688-3694, 1998.
Goldstein DS, Stull R, Eisenhofer G, and Gill JR. Urinary excretion of dihydroxyphenylalanine and dopamine during alterations of dietary salt intake in humans. Clin Sci (Colch) 76: 517-522, 1989.
Gomes P, Serrao MP, Viera-Coelho MA, and Soares-da-Silva P. Opossum kidney cells take up L -DOPA through an organic cation potential-dependent and proton-independent transporter. Cell Biol Int 21: 249-255, 1997.
Gomes P and Soares-da-Silva P. Na + -independent transporters, LAT-2 and b 0,+, exchange L -DOPA with neutral and basic amino acids in two clonal renal cell lines. J Membr Biol 186: 63-80, 2002.
Gonska T, Hirsh JR, and Schlatter E. Amino acid transport in the renal proximal tubule. Amino Acids (Vienna) 19: 395-407, 2000.
Haberle DA and Konigbauer B. Inhibition of tubuloglomerular feedback by the D1 agonist fenoldopam in chronically salt-loaded rats. J Physiol 441: 23-24, 1991.
Hagege J and Richet G. Proximal tubule dopamine histofluorescence in renal slices incubated with L -DOPA. Kidney Int 27: 3-8, 1985.
Hayashi M, Yamaji Y, Kitajima W, and Saruta T. Aromatic L -amino acid decarboxylase activity along the rat nephron. Am J Physiol Renal Fluid Electrolyte Physiol 258: F28-F33, 1990.
Jaeger P, Portmann L, Saunders A, Rosenberg LE, and Their SO. Anticystinuric effects of glutamine and of dietary sodium restriction. N Engl J Med 315: 1120-1123, 1986.
Jose P, Eisner GM, Drago J, Carey RM, and Felder RA. Dopamine receptor signaling defects in spontaneous hypertension. Am J Hypertens 9: 400-405, 1996.
Kim DK, Kanai Y, Matsuo H, Kim JY, Chairoungdua A, Kobayashi Y, Enomoto A, Cha S, Goya T, and Endou H. The human T-type amino acid transporter-1: characterization, gene organization, and chromosomal location. Genomics 79: 95-103, 2002.
Krishna GG, Danovitch GM, Beck FW, and Sowers JR. Dopaminergic mediation of the natriuretic response to volume expansion. J Lab Clin Med 105: 214-218, 1985.
Kuchel O and Shigetomi S. Defective dopamine generation from dihydroxyphenyl-alanine in stable essential hypertensive patients. Hypertension 19: 634-638, 1992.
McDonald RH, Goldberg LI, and McNay JL. Effect of dopamine in man: augmentation of sodium excretion, glomerular filtration rate and renal plasma flow. J Clin Invest 43: 1116-1124, 1964.
Oates NS, Ball SG, Perkins CM, and Lee MR. Plasma and urine dopamine in man given sodium chloride in the diet. Clin Sci (Colch) 56: 261-264, 1979.
Olsen NV, Hansen JM, Ladefoged SD, Fogh-Andersen N, and Leyssac PP. Renal tubular reabsorption of sodium and water during infusion of low dose dopamine in normal man. Clin Sci (Colch) 78: 503-507, 1990.
Peces R, Sanchez L, Gorostidi M, and Alvarez J. Effects of variation in sodium intake on cystinuria. Nephron 57: 421-423, 1991.
Rossier G, Meier C, Bauch C, Summa V, Sordat B, Verrey F, and Kuhn LC. LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of the kidney and intestine. J Biol Chem 274: 34948-34954, 1999.
Schnermann J, Todd KM, and Briggs JP. Effect of dopamine on the tubuloglomerular feedback mechanism. Am J Physiol Renal Fluid Electrolyte Physiol 258: F790-F798, 1990.
Schoors DF and Dupont AG. Increased dopamine-induced nephrogenous cAMP formation in hypertension. Am J Hypertens 4: 494-499, 1991.
Seri I, Kone BC, Gullans SR, Aperia A, Brener BA, and Ballermann BJ. Influence of sodium on dopamine-induced inhibition of renal cortical Na + -K + -ATPase. Am J Physiol Renal Fluid Electrolyte Physiol 258: F52-F60, 1990.
Shikuma R, Yoshimura M, Kambra S, Yamazaki R, Takahashi H, Takeda K, and Ijichi H. Dopaminergic modulation of salt-sensitivity in patients with essential hypertension. Life Sci 38: 915-921, 1986.
Soares-da-Silva P. Actin cytoskeleton, tubular sodium and the renal synthesis of dopamine. Biochem Pharmacol 3: 1883-1886, 1992.
Soares-da-Silva P. Enhanced protein kinase C mediated inhibition of renal dopamine synthesis during high sodium intake. Biochem Pharmacol 45: 1791-1800, 1993.
Soares-da-Silva P. Source and handling of renal dopamine: its physiological importance. News Physiol Sci 9: 128-134, 1994.
Soares-da-Silva P, Fernandes MH, and Pinto-do-O PC. Cell inward transport of L -DOPA and 3- O -methyl- L -DOPA in rat renal tubules. Br J Pharmacol 112: 611-615, 1994.
Soares-da-Silva P, Pestana M, and Fernandez MH. Involvement of tubular sodium in the formation of dopamine in the human renal cortex. J Am Soc Nephrol 3: 1591-1599, 1993.
Soares-da-Silva P, Serrao MP, and Viera-Coelho MA. Apical and basolateral uptake and intracellular fate of dopamine precursor L -dopa in LLC-PK1 cells. Am J Physiol Renal Physiol 274: F243-F251, 1998.
Sun D and Schafer JA. Dopamine inhibits AVP-dependent Na + transport and water permeability in rat CCD via a D4-like receptor. Am J Physiol Renal Fluid Electrolyte Physiol 271: F391-F400, 1996.
Takezako T, Noda K, Tsuji E, Koga M, Sasaguri M, and Arakawa K. Adenosine activates aromatic L -amino acid decarboxylase activity in the kidney and increases dopamine. J Am Soc Nephrol 12: 29-36, 2001.
Verrey F, Jack DL, Paulsen IT, Saier MH, and Pfeiffer R Jr. New glycoprotein-associated amino acid transporters. J Membr Biol 172: 181-192, 1999.
Wolfovitz E, Grossman E, Folio CJ, Keiser HR, and Kopin IJ. Derivation of urinary dopamine from plasma dihydroxyphenylalanine in humans. Clin Sci (Colch) 84: 549-557, 1993.
作者单位：Center of Mineral Metabolism and Clinical Research and Division of Nephrology, Department of Internal Medicine, University of Texas Southwestern Medical Center, and Medical Service, Department of Veterans Affairs Medical Center, Dallas, Texas 75390-8856