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(Hypertension. 2003;42:613.)
© 2003 American Heart Association, Inc.

Scientific Contributions

Organ-Specific Overexpression of Renal LAT2 and Enhanced Tubular L-DOPA Uptake Precede the Onset of Hypertension

Maria João Pinho; Pedro Gomes; Maria Paula Serrão; Maria João Bonifácio; Patrício Soares-da-Silva

From the Institute of Pharmacology and Therapeutics, Faculty of Medicine, Porto, Portugal.

Correspondence to P. Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200 Porto, Portugal. E-mail psoaresdasilva{at}netcabo.pt

	Abstract

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Spontaneously hypertensive rats (SHR) might have increased renal production of dopamine. L-3,4-Dihydroxyphenylalanine (L-DOPA) uptake in renal epithelial cells is promoted through the type 2 L-type amino acid transporter (LAT2), and this might rate-limit the synthesis of renal dopamine. The present study evaluated L-DOPA uptake in isolated renal proximal tubules of SHR and normotensive controls (Wistar-Kyoto rats [WKY]). Expression of LAT1 and LAT2 in the renal cortex and intestinal mucosa was also evaluated. Tubular uptake of L-DOPA in WKY and SHR was a saturable process, being greater in the latter than the former at both 4 and 12 weeks of age. cDNA fragments (LAT1, 688 bp; LAT2, 729 bp) labeled with ³²P were used as probes for Northern blot analysis. Expression of LAT2 in SHR kidneys was higher than in WKY kidneys. This increase was more marked at 4 than at 12 weeks of age. Intestinal LAT2 expression, however, was identical in SHR and WKY at both 4 and 12 week of age. By Northern blot analysis, the LAT1 transcript was not identified in either the kidney or intestine. Kidney total RNA was then reverse-transcribed and amplified by polymerase chain reaction with specific primers for LAT1. The presence of a fragment of the expected size for LAT1 led to the conclusion that LAT1 mRNA is a rare message in kidney. We conclude that overexpression of LAT2 in the SHR kidney might contribute to the enhanced L-DOPA uptake, which is organ specific and precedes the onset of hypertension.

Key Words: gene expression • dopamine • rats, spontaneously hypertensive • hypertension, renovascular • kidney

	Introduction

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Dopamine of renal origin exerts natriuretic and diuretic effects by activating D₁-like receptors located at various regions in the nephron.^1,2 At the level of the proximal tubule, the overall increase in sodium excretion produced by dopamine and D₁ receptor agonists results from inhibition of the main sodium transport mechanisms at the basolateral and apical membranes, respectively, Na⁺,K⁺-ATPase^3,4 and the Na⁺/H⁺ exchanger.⁵ In the spontaneously hypertensive rat (SHR), dopamine D₁-like receptor–mediated natriuretic and diuretic responses are diminished under normal conditions as well as during short-term volume expansion (5% body weight) compared with those in normotensive, control, Wistar-Kyoto rats (WKY).^6–8 However, dopamine production and excretion in SHR are normal or even increased when compared with those in WKY,^9–13 although this pattern might correspond to an age-dependent process. This would suggest that in SHR, the increased ability to form dopamine at the kidney level might correspond to an attempt to overcome the deficient dopamine-mediated natriuresis,⁸ as has been reported in aged Fischer 344 rats.¹⁴ The proximal tubules, but not distal segments of the nephron, are endowed with high aromatic L-amino acid decarboxylase (AADC) activity, and epithelial cells of proximal tubules have been demonstrated to synthesize dopamine from circulating or filtered L-3,4-dihydroxyphenylalanine (L-DOPA).^4,15,16 Recently, L-DOPA uptake in renal epithelial cells was found to be promoted through the Na⁺-independent and pH-sensitive type 2 L-type amino acid transporter (LAT2),¹⁷ the activity of which might rate-limit the synthesis of renal dopamine.¹⁸

The hypothesis we explored in the present study concerns the occurrence of adaptations in renal L-DOPA transporters in hypertension and their putative role in enhanced renal dopamine formation in genetic hypertension. For this purpose, we measured L-DOPA uptake in isolated renal proximal tubules and evaluated the presence and expression of LAT1 and LAT2 in the renal cortex and intestinal mucosa of SHR and WKY at 4 and 12 weeks of age. The activity of renal AADC was also measured to assess the ability of renal tissues to convert intracellular L-DOPA to dopamine.

	Methods

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Animals and Preparation of Renal Tubules
Renal tubules from male SHR and WKY (Harlan-Inferfauna, Barcelona, Spain) were prepared by following the techniques previously described and were found to contain predominantly proximal tubules.^19–21 The viability of proximal renal tubules used in this study was assessed by the trypan blue (0.2% wt/vol) exclusion method.¹⁹ Under these conditions, >90% of the renal tubules excluded the dye.

Tubular Uptake Studies
The nonsaturable component of L-DOPA uptake was determined in experiments conducted at 4°C. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA taken up into the renal tubules at 37°C minus the values obtained for the nonsaturable component at 4°C, as previously described.^19–21 Incubation was stopped by cooling, and an aliquot (300 µL) of the incubation medium containing the renal tubules was used for the assay of L-DOPA by high-performance liquid chromatography with electrochemical detection.^19–21

AADC Activity
AADC activity was determined in homogenates of renal tissues with L-DOPA (10² to 10⁴ µmol/L) as the substrate, as previously described.^22,23

RT-PCR
Polymerase chain reaction (PCR) was performed with the use of a commercially available system (Platinum TaqPCRx DNA polymerase, Invitrogen) with 1x enhancer (LAT1) or without enhancer (LAT2). Amplification conditions were as follows: hot start of 2 minutes at 95°C; 30 cycles of denaturing (95°C for 30 seconds), annealing (58°C for 30 seconds), and extension (68°C for 45 seconds); and a final extension of 7 minutes at 68°C.

cDNA Probe Generation
Kidney total RNA extracted from a Wistar rat was reverse-transcribed (RT) and amplified by PCR with use of a commercially available (Titan RT-PCR kit, Roche) and rat LAT1 (GenBank accession No. AF104032) and rat LAT2 (GenBank accession No. NM_053442 and GenBank accession No. AF171669) primer sets. PCR conditions were as follows: hot start of 2 minutes at 94°C; 10 cycles of denaturing (94°C for 30 seconds), annealing (55°C for 1 minute), and extension (68°C for 1 minute); 15 cycles of denaturing (94°C for 30 seconds), annealing (55°C for 45 seconds), and extension (68°C for 45 seconds); and a final extension of 5 minutes at 68°C. The products were gel-purified (Qiaex II, Qiagen) and reamplified with DNA polymerase (PfuTurbo, Stratagene) under PCR conditions similar to those described earlier, except that the extension step was performed at 72°C. The resultant products were cloned into vectors (pZErO-1 for LAT1 and pCR-Blunt II-Topo for LAT2) with the use of cloning kits (Zero Background and Zero Blunt TOPO PCR, respectively; Invitrogen).

Northern Blot Analysis
Total RNA was fractionated on formaldehyde-agarose gels (1% Seakem agarose, 2.2 mol/L formaldehyde, and 1x MOPS). Samples were mixed with loading buffer (1.6 mol/L formaldehyde, 0.36 mg/mL bromophenol blue, 7% glycerol, and 1x MOPS) and heated at 65°C for 5 minutes before being loaded. Gels were run at a constant voltage of 70 V for 3 hours. After being washed, RNA was transferred to positively charged nylon membranes (Roche) by alkaline downward capillary elution with 5x standard saline citrate (SSC)/0.1% NaOH. The RNA was then UV–cross-linked (UVC500 crosslinker, Amersham Biosciences).

LAT1 and LAT2 probes were labeled by random-primer labeling with use of a kit (Random Prime Labeling and [-³²P]dCTP, both from Amersham Biosciences). The denatured probes were added to the hybridization buffer and incubated with the blots at 65°C for 3 hours. After hybridization, the blots were washed twice in 2x SSC/0.1% sodium dodecyl sulfate (SDS) for 10 minutes at room temperature; twice in 1x SSC/0.1% SDS for 15 minutes at 65°C; and finally, once in 0.1x SSC/0.1% SDS for 15 minutes at 65°C. Northern blots were exposed to a phosphorimaging screen (Bio-Rad) and visualized with an imager (Personal Molecular Imager FX, Bio-Rad). Blots were stripped and rehybridized with a rat ß-actin probe²⁴ to correct for variances between samples.

Statistics
Results are mean±SEM of values for the indicated number of determinations. V_max and K_m values for the uptake and decarboxylation of L-DOPA were calculated from nonlinear regression analysis (GraphPad Prism statistics software package).²⁵ Geometric means are given with 95% confidence limits, and arithmetic means are given with SEM. Statistical analysis was performed by 1-way ANOVA, followed by Student t test for unpaired comparisons. A probability value <0.05 was assumed to denote a significant difference.

	Results

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Blood pressure (systolic, SBP; diastolic, DBP) and heart rate were measured in conscious, restrained animals between 7 and 10 AM with a photoelectric tail-cuff pulse detector (LE 5000, Letica). At 4 weeks of age, body weight (BW, in grams), SBP, and DBP (both in mm Hg) did not differ between WKY (BW=91±4, SBP=115±7, DBP=74±6) and SHR (BW=96±7, SBP=118±9, DBP=76±5). At 12 weeks of age, both SBP and DBP were significantly (P<0.05) higher in SHR (SBP=191±4, DBP=115±2) than in WKY (SBP=139±3, DBP=79±2), whereas BW did not differ between SHR (BW=303±14) and WKY (BW=277±9).

Tubular Uptake of L-DOPA
Incubation of renal tubules at 4°C in the presence of increasing concentrations of L-DOPA resulted in a concentration-dependent accumulation of the substrate.¹⁹ Benserazide (10 µmol/L, Sigma Chemical Co) and tolcapone (1 µmol/L; kindly donated by the late Prof Mosé Da Prada, Hoffman–La Roche, Basel, Switzerland) were also added to the Hanks’ medium to inhibit the enzymes AADC and catechol-O-methyltransferase, respectively.¹⁹ When the experiments were performed at 37°C, accumulation of L-DOPA in the renal tubules was greater than that which occurred at 4°C and showed a trend for saturation, as previously reported.^{19,21,23,26–28} In the first series of experiments, we evaluated the accumulation of L-DOPA in renal tubules from 4- and 12-week-old WKY and SHR incubated with a nonsaturating concentration of L-DOPA (30 µmol/L). Accumulation of L-DOPA at 37°C was greater than that which occurred at 4°C (data not shown). The temperature-sensitive component of L-DOPA accumulation in 4- and 12-week-old SHR was significantly greater than that observed in corresponding aged-matched WKY (Figure 1). However, the difference between SHR and WKY in terms of the temperature-sensitive component of L-DOPA accumulation was more evident at 4 weeks than at 12 weeks (Figure 1). Next, we evaluated the kinetics of L-DOPA transport in renal tubules from 4-week-old SHR and WKY. For this purpose, renal tubules were incubated with increasing concentrations of L-DOPA (3 to 10³ µmol/L) at 37°C and 4°C. The saturable component of L-DOPA uptake was derived from the total amount of L-DOPA accumulated in renal tubules at 37°C minus the values obtained in experiments conducted at 4°C. As shown in Figure 2, the saturable component of L-DOPA uptake in renal tubules from SHR was significantly higher than that observed in WKY (see also Table 1). No significant difference was observed between K_m values for the saturable component of L-DOPA uptake in WKY and SHR (Table 1). The diffusion rate of transfer of L-DOPA was found to be similar in WKY and SHR (Table 1). It should be underlined that evaluation of specific L-DOPA uptake into renal tubules based on differences between fluxes at 37°C and 4°C has limitations, namely, on the extent of passive diffusion through the lipid portion of the membrane that might be altered at low temperature. However, our previous experience in suspensions of renal tubules indicates that in analysis of nonspecific uptake or cell binding of L-DOPA determined at 4°C with the competitive inhibitors 3-O-methyl-L-DOPA¹⁹ and L-5-hydroxytryptophan,²⁰ the uncoupling agent 2,4-dinitrophenol²¹ provides identical values for the saturable uptake of L-DOPA.

View larger version (14K): [in this window] [in a new window]   Figure 1. Tubular uptake of L-DOPA (100 µmol/L) in isolated renal tubules from SHR and WKY at 4 and 12 weeks of age. Columns represent means of 4 determinations per group; vertical lines indicate SEM. *P<0.05, SHR vs WKY.

View larger version (20K): [in this window] [in a new window]   Figure 2. Concentration-dependent tubular uptake of L-DOPA (3 to 103 µmol/L) in isolated renal tubules from SHR and WKY at 4 weeks of age. Symbols represent means of 4 determinations per group; vertical lines indicate SEM.

View this table: [in this window] [in a new window]   TABLE 1. Kinetic Parameters for the Uptake of L-DOPA in Isolated Renal Tubules From SHR and WKY at 4 Weeks of Age

Renal AADC Activity
In experiments performed in homogenates of renal tubules, decarboxylation of L-DOPA to dopamine was found to be dependent on the concentration of L-DOPA used (10² to 10⁴ µmol/L). No significant difference was observed between the V_max values of renal AAAD in WKY and SHR at both 4 and 12 weeks of age (Table 2). K_m values for AADC were also found not to differ between WKY and SHR at both 4 and 12 weeks of age (Table 2).

View this table: [in this window] [in a new window]   TABLE 2. Kinetic Parameters of Aromatic AADC Activity in Homogenates of Isolated Renal Tubules From SHR and WKY

Detection of LAT1 and LAT2 Transcripts in WKY and SHR
The presence of LAT1 and LAT2 transcripts in kidney cortexes and intestinal mucosa of 12-week-old WKY and SHR animals was first examined by RT-PCR with specific primers for either LAT1 or LAT2 rat cDNA sequences. The LAT1 cDNA was amplified by PCR with 2 sets of primers: 1 specific for rat LAT1 (forward, 5'-CAT CAT CGG TTC GGG CAT CTT-3'; reverse, 5'-CAG GGT GAC AAT GGG CAA GGA-3') and another simultaneously specific for human, rat, and mouse LAT1 (forward, 5'-GG(C/T) TCG (G/T)GC ATC TTC GT-3'; reverse, 5'-(G/A)CA (G/C)AG CCA GTT GAA GAA GC-3'), corresponding to nucleotides 285 and 1267 of the human cDNA (GenBank accession No. AF104032). The LAT2 cDNA was amplified by PCR with 1 set of primers simultaneously specific for human, rat, and mouse LAT2 (forward, 5'-CA(C/G) CCG (A/G)AC AAC ACC G(A/C)(A/C/G) AAG-3'; reverse, 5'-TGC CAG TA(A/G) ACA CCC AGG AA(A/G)-3'), corresponding to nucleotides 242 and 1612 of the human cDNA (GenBank accession No. AF171669). The expected 688-bp fragment corresponding to LAT1 was identified in total RNA from the kidneys and intestinal mucosa of both SHR and WKY (Figure 3). The 1391-bp product was also obtained when the same RNA samples were subjected to RT and amplified by PCR with the LAT2 rat–specific primers in both SHR and WKY kidney RNA and intestinal epithelium RNA (Figure 3).

View larger version (45K): [in this window] [in a new window]   Figure 3. Detection of LAT1 and LAT2 in kidney and intestine total RNA extracted from WKY and SHR aged 12 weeks with LAT1 and LAT2 rat-specific primers. MW indicates molecular weight.

Expression of LAT1 and LAT2
Expression of LAT2 mRNA in the kidney and intestinal mucosa of 4- and 12-week-old SHR and WKY was studied by Northern blot analysis. Kidney total RNA extracted from a Wistar rat was subjected to RT and amplified by PCR with use of a kit (Titan RT-PCR, Roche), rat LAT1 (described earlier) and rat LAT2 forward (5'-AGG CCC ATG GTC AAG GTC AGT GC-3', and rat LAT2 reverse (5'-CCA GGG GAG GAA AGA GGA GGA CG-3'; based on the published cDNA sequence [GenBank accession No. NM_053442]) primer sets. The 729-bp LAT2 cDNA probe hybridized with a transcript of 4 kb, the size of the rat LAT2 primary transcript (4117 bp). The LAT2 transcript appeared to be strongly expressed in SHR kidneys compared with WKY kidneys, and this increase was greater in the 4-week-old than the 12-week-old animals (Figure 4). In the intestinal mucosa, no significant differences were observed in the expression of LAT2 mRNA in either SHR or WKY, both at 4 and 12 weeks of age (Figure 5). As for LAT1, it was not possible to identify the 3-kb transcript by Northern blot analysis in either kidney or intestine, suggesting that if it is expressed, it is below the sensitivity of the Northern blot.

View larger version (21K): [in this window] [in a new window]   Figure 4. Kidney LAT2 mRNA quantification. A, Northern blot of 10 µg total RNA from 4- and 12-week-old WKY and SHR kidneys. RNA was hybridized to rat LAT2 cDNA fragment. B, Graph representing LAT2–ß-actin mRNA ratios for n=3 or 4. Significantly different from WKY (*P<0.05) and corresponding values at 4 weeks (#P<0.05).

View larger version (25K): [in this window] [in a new window]   Figure 5. Intestinal LAT2 mRNA quantification. A, Northern blot of 10 µg total RNA from 4- and 12-week-old WKY and SHR intestine. RNA was hybridized to rat LAT2 cDNA fragment. B, Graph representing LAT2–ß-actin mRNA ratios for n=3 or 4. Significantly different corresponding values at 4 weeks (#P<0.05).

	Discussion

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The results presented herein show that the tubular uptake of L-DOPA in WKY and SHR was a saturable process, being higher in the latter than the former at both 4 and 12 weeks of age. Expression of LAT2 in SHR was higher than in WKY kidneys. This increase was more marked at 4 than at 12 weeks of age. Expression of intestinal LAT2 was identical in SHR and WKY at both 4 and 12 weeks of age. The Northern blot analysis did not detect LAT1 mRNA in either the kidney or intestine. Kidney total RNA was then subjected to RT and amplified by PCR with specific primers for LAT1. The presence of a fragment of the expected size for LAT1 led to the conclusion that LAT1 mRNA is a rare message in kidney. It is suggested that overexpression of LAT2 in the SHR kidney might contribute to the enhanced L-DOPA uptake, which is organ specific and precedes the onset of hypertension.

Although the kidney is endowed with 1 of the highest levels of aromatic AADC activities in the body and plasma levels of L-DOPA are in the nanomoles per milliliter range,^29,30 the rate-limiting step for the synthesis of dopamine in renal tissues is still a matter of debate. Because K_m values for L-DOPA uptake are 10 times lower than the K_m values for decarboxylation of L-DOPA, it could be possible that L-DOPA uptake rather than decarboxylation might limit the rate of formation of dopamine. However, the transporters involved in the uptake of L-DOPA by renal epithelial cells have not been clearly identified. At present, candidate transport systems for L-DOPA might 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 epithelium³¹ and the latter in mouse brain capillary endothelial cells.³² This conflicts with the view that LAT1-specific mRNA is expressed in most human tissues, with the notable exception of the intestine.³³ On the other hand, the mRNA corresponding to LAT2 examined by Northern blot analysis was strongly expressed in the small intestine.^34,35 In agreement with the view that LAT2 might play a role in L-DOPA transport is the recent observation that in opossum kidney (OK) renal tubular cells, L-DOPA uses at least 2 major transporters, systems LAT2 and b^0,+.¹⁷ The transport of L-DOPA by LAT2 corresponds to an Na⁺-independent transporter with a broad specificity for small and large, neutral amino acids that is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid.¹⁷ The transport of L-DOPA by system b^0,+ is an Na⁺-independent transporter for neutral and basic amino acids that also recognizes the diamino acid cystine.¹⁷ The results presented herein agree with the view that LAT2 rather than LAT1 might be involved in the renal transport of L-DOPA, owing to the low level of expression of the latter transporter. Major differences between LAT1 and LAT2 are concerned with amino acid specificity and affinity. The affinity of LAT1 for large, neutral amino acids is higher than that of LAT2. LAT1 also transports small, neutral amino acids, such as L-alanine, glycine, and L-cysteine,³⁶ though with a lower affinity to substrate amino acids than that of LAT2.³⁵ The K_m values reported here for the uptake of L-DOPA into SHR and WKY renal proximal tubules are within the range described for amino acid transport by LAT2.^35,36 On the other hand, the enhanced expression of LAT2 in the renal cortex of 4-week-old SHR is correlated positively to the increased ability of renal tubules to take up L-DOPA when compared with the aged-matched, normotensive WKY; this trend was less evident at 12 weeks of age. Because dopamine production and excretion in SHR are normal or even increased when compared with those in WKY,^9–13 the enhanced tubular uptake of L-DOPA and the overexpression of LAT2 in SHR might correspond to an attempt to overcome the deficient dopamine-mediated natiuresis.^6–8 In this respect, it is interesting to emphasize the fact that in the SHR, stimulation of D₁-like receptors also fails to inhibit intestinal Na⁺,K⁺-ATPase activity, and dopamine levels in the intestinal mucosa of SHR do not differ from those in WKY.³⁷ Therefore, it might be suggested that at the intestinal level, there is no adaptation to produce more dopamine, despite the failure of dopaminergic mechanisms to inhibit intestinal Na⁺,K⁺-ATPase activity.

The overexpression of LAT2 and the enhanced uptake of L-DOPA in the SHR are, however, not hypertension-related phenomena, because they precede the onset of hypertension, though being organ specific. It is possible that the renal microenvironment of the SHR might contribute to the overexpression of LAT2 and the enhanced uptake of L-DOPA rather than the increase in blood pressure. In fact, it has been suggested that salt-sensitive–hypertensive patients take up less L-DOPA and synthesize less dopamine at the kidney level,^38,39 whereas SHR appear to be endowed with an enhanced ability to take up L-DOPA and Na⁺.^10,12,13 Another important aspect is concerned with the fact that Na⁺ is a powerful stimulus for the production of renal dopamine in humans and some laboratory animals,⁴⁰ and L-DOPA uptake in the human kidney is an Na⁺-dependent and ouabain-sensitive process.⁴¹ The finding that L-DOPA uptake in renal (LLC-PK₁ and OK cells)^17,27 and nonrenal (RBE4, DITNC1, and Neuro 2A)^42,43 cultured cells is largely promoted through the Na⁺-insensitive, pH-dependent, and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid–sensitive LAT2 transporter contrasts with the role of Na⁺ in the formation of renal dopamine.⁴⁰ In these cell types, most of the L-DOPA enters the cells in an Na⁺-independent manner; only a minor component of L-DOPA uptake (15%) was found to require extracellular Na⁺. This was particularly evident in OK_HC cells,¹⁷ which, like the renal tubular cells from hypertensive subjects,⁴⁴ are endowed with an enhanced ability to transport Na⁺ through the Na⁺/H⁺ exchanger and Na⁺,K⁺-ATPase.^43,44

Perspectives
In the SHR and in some forms of human hypertension, despite a normal dopamine receptor density, there is defective transduction of the D₁ receptor signal in renal proximal tubules.⁸ This coupling defect is genetic (precedes the onset of hypertension and cosegregates with the hypertensive phenotype), is receptor specific (not shared by other humoral agents), and is organ and nephron-segment selective (occurs in proximal tubules but not in cortical collecting ducts or the brain striatum). A consequence of the defective dopamine receptor/adenylyl cyclase coupling in the SHR and hypertensive subjects is a decreased ability to promote natriuresis. We have demonstrated for the first time that overexpression of LAT2 in the SHR kidney is organ specific and precedes the onset of hypertension, this being accompanied by an enhanced ability to take up L-DOPA. Therefore, we suggest that overexpression of renal LAT2 might constitute the basis for the enhanced renal production of dopamine in the SHR in an attempt overcome the deficient dopamine-mediated natriuresis generally observed in this genetic model of hypertension. This adaptive mechanism might be limited to renal tissues, because at the intestinal level where defective transduction of the D₁ receptor signal also occurs, it is accompanied by increases in neither dopamine tissue levels nor intestinal LAT2 expression.

	Acknowledgments

 
This study was supported by grant POCTI/SAU/35747/2000 from the Fundação para a Ciência e a Tecnologia.

	Footnotes

 
M.J.B. is currently affiliated with the Department of Research and Development, BIAL, Mamede do Coronado, Portugal.

Received March 18, 2003; first decision April 17, 2003; accepted August 7, 2003.

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