This study assessed the role of adrenergic receptors on the regulation of the uptake of l-dopa and the production of dopamine by renal tubular cells. Scatchard analysis showed two l-dopa uptake sites with different affinities (K m 0.316 vs 1.53 μM). l-Dopa uptake was decreased by the nonselective adrenergic agonists epinephrine or norepinephrine (40%), by the β-selective agonist isoproterenol or the β2-selective agonist terbutaline (60%), but not by α-selective agonists (all 1 μM). The effect of norepinephrine, isoproterenol, or terbutaline was unaffected by addition of the β1-antagonist atenolol, abolished by ICI-118,551, a β2-antagonist (both 0.1 μM), and mimicked by the addition of dibutyryl-cAMP (1 μM). Preincubation with terbutaline decreased the number of high-affinity uptake sites (V max = 1.10 ± 0.3 vs. 0.5 ± 0.1 pmol · mg protein−1 · min−1) without changing their affinity. Norepinephrine or terbutaline decreased dopamine production by isolated cells, and this effect was abolished by ICI-118,551 (0.1 μM). In vivo administration of ICI-118,551 reduced the urinary excretion of l-dopa and increased the excretion of 3,4-dihydroxyphenylacetic acid without significant changes in plasma l-dopa concentrations. These results demonstrate that stimulation of β2-adrenergic receptors decreases the number of high-affinity l-dopa uptake sites in isolated tubular cells resulting in a reduction of the uptake of l-dopa and the production of dopamine and provide evidence for the presence of this mechanism in the intact animal.
- cellular transport
l-3,4-dihydroxyphenylalanine(l-dopa) is the immediate product of the rate-limiting step in catecholamine biosynthesis and the precursor of all the endogenous catecholamines (15). Circulating l-dopa is removed by the kidney, and a major proportion of dopamine in rat and human urine derives from the renal decarboxylation in proximal tubular cells of l-dopa reabsorbed from ultrafiltrated plasma (17, 20, 29). Several lines of evidence indicate that renal dopamine plays an important role in the regulation of salt balance acting locally as an autocrine-paracrine substance (1, 11, 20). Renal production of dopamine is dependent on substrate availability, on the uptake of l-dopa into tubular epithelial cells, and on the activity of aromatic l-amino acid decarboxylase (AADC), the enzyme responsible for the conversion of l-dopa into dopamine. Several studies performed in renal tissues have failed to demonstrate parallel changes in the activity of AADC and the production of dopamine and suggested that the uptake ofl-dopa is the rate-limiting process in dopamine formation (2, 20).
It is well established that the nephron reabsorbs l-amino acids almost completely from the glomerular filtrate and that the reabsorption occurs mainly in the proximal tubule (7,30). Early studies have demonstrated that the reabsorption of l-dopa in the proximal convoluted tubules is an active process, independent of the AADC activity, with great structural specificity (6). In a previous study, using brush-border membrane vesicle preparations, we have characterized the transport ofl-dopa as an Na+-dependent cotransport, which is impaired in aged rats (2). However, only scant biochemical detail is available on the transport of l-dopa and of other l-amino acids in the kidney, and the regulation of this process is still poorly understood.
The adrenergic nervous system modulates several aspects of kidney function. The kidney has large numbers of unevenly distributed adrenergic receptors, and it has been suggested that these receptors may subserve specific functions in the different kidney zones. Adrenergic receptors of the α1-, α2-, β1-, and β2-subtypes have been characterized in rat renal proximal tubules (22,27) and have been shown to be involved in the modulation of tubular transport (19, 23,24). In tissues other than the kidney, adrenergic receptors have been shown to be implicated in the regulation of the transport of sugars (4, 5, 13) and amino acids (10, 21).
This study was aimed to assess the role of adrenergic receptors on the regulation of the uptake of l-dopa and therefore on the production of dopamine by renal tubular cells. The present results show that the uptake of l-dopa by isolated rat renal tubular cells is decreased by β2-adrenergic stimulation but not affected by α- or β1-receptor stimulation. Subsequently, to provide evidence for the presence of this regulatory mechanism in in vivo conditions, we assessed the effects of administration of a β2-adrenergic receptor antagonist to intact animals.
MATERIALS AND METHODS
l-Dopa, collagenase (type IV), DMEM, norepinephrine, epinephrine, N,2′-O-dibutyryl-cAMP, 3-methoxy-dl-tyrosine, isoproterenol, terbutaline, methoxamine, atenolol, and phentolamine, were obtained from Sigma Chemical (St. Louis, MO); 3-hydroxybenzylhydrazine (an AADC inhibitor), prazosin, and ICI-118,551 (a selective β2-antagonist) were obtained from Research Biochemicals International (Natick, MA); and octopamine was obtained from Calbiochem (San Diego, CA).
Three-month male Wistar rats weighing between 250 and 300 g were used in this study. All animals were inbred in our laboratory, kept at 22°C on a 12:12-h dark-light cycle, and given free access to normal rat diet (protein content 25%) and tap water.
Preparation of proximal tubule cell suspension.
Proximal tubule cells suspensions were prepared as previously described (3). Briefly, rats were killed by decapitation, and the kidneys were rapidly removed and rinsed free from blood with saline (0.9% wt/vol NaCl solution). The kidneys were placed on an ice-cold glass plate, and the cortex was isolated. Thereafter, the tissue was minced on ice to a paste-like consistency. The cortical tissue was digested with 0.7 mg/ml collagenase (type IV, Sigma) in 10 ml DMEM supplemented with 20 mM HEPES and 24 mM NaHCO3 (pH 7.4). Incubation was carried out in a shaking water bath at 37°C for 30 min in an atmosphere of 95% O2-5% CO2. It was cooled on ice and poured through graded sieves (180, 75, 53, and 38 μm in pore size) to obtain a cell suspension. This suspension contains mostly proximal tubular cells (3). To separate the remaining blood cells and traces of collagenase, the cell suspension was centrifuged at 400 rpm for 4 min and washed, and the final pellet was resuspended in Krebs buffer. Each determination was performed using 50 μl of the cell suspension containing 150–250 μg protein. The cells were used within 2 h of preparation and kept on ice until studied. The quality of each preparation was monitored by microscopy, and the viability was assessed by Trypan blue exclusion. Protein concentration was measured by the method of Lowry, with bovine serum albumin as a standard.
Transport of l-dopa into isolated proximal tubular cells.
The transport of l-dopa was determined using a modification of a previously described method (2). Briefly, cells were preincubated for 20 min in Krebs buffer (in mM: 120 NaCl, 4.7 KCl, 1.2 MgSO4, 2.4 CaCl2, 24 NaHCO3, 1.2 KH2PO4, 0.5 EDTA, 0.57 ascorbic acid, and 11 glucose, pH 7.4) with the addition of 3-hydroxybenzylhydrazine (250 μM) to inhibit the AADC activity, in the absence or presence of 3-methoxy-l-tyrosine (3-O-methyldopa, 100 μM), isoproterenol, norepinephrine, epinephrine, terbutaline, octopamine, methoxamine (all 1 μM), atenolol, or ICI-118,551 (both 0.1 μM), or increasing concentrations of isoproterenol or dibutyryl-cAMP (both 1 nM to 1 μM). In experiments performed to test the influence of Na+ on l-dopa uptake, cells were incubated in Krebs buffer (144 mM Na+, 6 mM K+) or in lower Na+ Krebs buffer (44 mM Na+, 106 mM K+). l-Dopa uptake was started by the addition of the indicated concentrations ofl-dopa to the incubation medium. Unless specified, the preincubation and incubation reactions were carried out for 20 min at room temperature. During preincubation and incubation, cells were continuously shaken. Reactions were stopped by centrifugation (4°C, 600 rpm, 3 min) and rapid removal of uptake solution by means of a vacuum pump connected to a Pasteur pipette followed by immediate rinsing twice with ice-cold Krebs solution and centrifuged (4°C, 600 rpm, 3 min). The pellet was resuspended in 200 μl of 0.3 N HClO4, disrupted (Sonifier Cell Disruptor, model w185; Heat Systems-Ultrasonics), and stored at −20°C until assayed.
Production of dopamine by isolated proximal tubular cells.
In experiments aimed to study the accumulation of newly formed dopamine in the incubation medium, isolated tubular cells were preincubated in the same conditions as described above, with the exception that the AADC inhibitor was not added. Cells were preincubated in the absence or presence of norepinephrine or terbutaline (both 1 μM), or atenolol or ICI-118,551 (both 0.1 μM). Reactions were started by incubation with l-dopa (200 nM, 20 min) and were stopped by centrifugation (4°C, 600 rpm, 3 min). The incubation medium was immediately collected on 200 μl of 0.3 N HClO4 and stored at −20°C until assay.
Urine and plasma collection.
On the day of the study, rats were given ICI-118,551 (1.5 mg/kg ip, twice, every 12 h starting at 9.00 AM) or vehicle and placed in metabolic cages for 24-h urine collection. Urine was collected into 100-ml polyethylene tubes containing 500 μl of 6 N HCl. Samples were stored at −20°C until assayed for catechols and creatinine. Blood samples were obtained from the tail from vehicle or ICI-118,551-treated animals. Plasma was immediately separated from blood cells by centrifugation (6,000 rpm, 10 min) and stored frozen until assayed forl-dopa concentration.
The catechols in 20-μl aliquots of urine, 250 μl of plasma, 200 μl of cell homogenates, or 500 μl of incubation medium were determined as reported previously (2). Briefly, catechols in the samples were partially purified by batch alumina extraction, separated by reverse-phase high-pressure liquid chromatography using a 4.6 × 250-mm Zorbax RxC18 column (DuPont) and quantified amperometrically by the current produced upon exposure of the column effluent to oxidizing and then reducing potentials in series using a triple-electrode system (ESA, Bedford, MA). Recovery through the alumina extraction step averaged 70–80% for dopamine, 45–55% for l-dopa and 3,4-dihydroxyphenylglycol (DHPG) and 40% for 3,4-dihydroxyphenylacetic acid (DOPAC). Catechol concentrations in each sample were corrected for recovery of an internal standard, dihydroxybenzylamine. Levels of l-dopa, DHPG, and DOPAC were further corrected for differences in recovery of the internal standard and of these catechols in a mixture of external standards. The limit of detection was about 15 pg/volume assayed for each catechol.
Data are means ± SE. Statistical comparisons between two groups were done by Mann-Whitney U Test. Comparisons among several groups were done by analysis of variance (ANOVA) followed by Newman-Keuls test. P < 0.05 defined statistical significance. The lines of the Scatchard plots were determined by linear regression, and the coefficients of linear regression (r) were 0.80 or better in all cases.K m and V max were calculated from Scatchard plots.
Figure 1 shows the time dependency of the uptake of l-dopa into isolated rat renal tubular cells. In our experimental conditions, intracellular l-dopa content reached maximal levels after 15-min incubation. Figure 1 also shows that the addition of 100 μM 3-O-methyldopa, an amino acid structurally similar to l-dopa, to the incubation medium reduced by 90% the uptake of l-dopa into isolated cells.
The uptake of l-dopa by isolated cells was reduced by 37 ± 7% when the Na+ concentration in the incubation medium was decreased from 144 to 44 mM.
Figure 2 shows that the uptake ofl-dopa into cells in the presence of increasing concentrations of l-dopa (100 nM to 100 μM) in the incubation medium was concentration dependent, reaching its maximal levels at 10 μM l-dopa. Scatchard analysis of the saturation curve showed two different sites for the uptake ofl-dopa by these cells, one site with high affinity (K m = 316 nM) and aV max of 1.22 pmol · mg protein−1 · min−1, and a second site with a lower affinity (K m = 1.53 μM) and a V max of 2.45 pmol · mg protein−1 · min−1 (Fig.3).
Preincubation of the cells with the nonselective adrenergic agonists norepinephrine or epinephrine (1 μM) decreased l-dopa uptake by 40% (P < 0.02). The same concentration of the β-selective agonists isoproterenol or terbutaline reducedl-dopa uptake by 60% (P < 0.01). On the other hand, the α-selective agonists methoxamine or octopamine (1 μM) had no significant effect. The effect of norepinephrine and epinephrine on l-dopa uptake was not affected by the presence of the α-adrenergic antagonists phentolamine or prazosin (0.1 μM) (Table 1).
Preincubation of the cells with different concentrations (1 nM to 1 μM) of isoproterenol demonstrated that the inhibitory effect of the β-selective agonist on l-dopa uptake was dose dependent (Fig. 4).
The effect of 1 μM isoproterenol, terbutaline, or norepinephrine was not reversed by the presence of the β1-adrenergic antagonist atenolol (0.1 μM) in the incubation medium but was completely abolished by 0.1 μM ICI-118,551, a β2-adrenergic antagonist (Fig.5).
The effect of 1 μM terbutaline was tested at different concentrations of l-dopa in the medium (100 nM to 1 μM). Scatchard analysis of the saturation curves in the absence and presence of terbutaline showed a clear decrease in the number of high-affinity uptake sites (1.10 ± 0.28 vs. 0.52 ± 0.06 pmol · mg protein−1 · min−1; P< 0.02) with no changes in the K m (416 ± 42 vs. 407 ± 38 nM) (Fig. 6). A time course study of the effect of addition of terbutaline to the incubation medium showed that the maximal inhibitory effect was achieved at 20 min (Fig. 7).
Addition of increasing concentrations (1 nM to 1 μM) of the cAMP analog dibutyryl-cAMP (dcAMP) also decreased, dose dependently,l-dopa uptake (Table 2).
As expected, the incubation of cells with l-dopa (200 nM to 10 μM) produced a dose-dependent increase of dopamine in the medium (Fig. 8). The production of dopamine by cells incubated with l-dopa (200 nM) was decreased (P < 0.04) by preincubation with either the nonselective adrenergic agonist norepinephrine or the β2-selective agonist terbutaline (both at 1 μM). The decrease of dopamine production elicited by both norepinephrine and terbutaline were not significantly affected by atenolol but were antagonized by the presence in the medium of ICI-118,551 (Table3).
Administration of ICI-118,551 to intact animals (1.5 mg/kg, twice a day, 24 h) reduced significantly (P < 0.05) the urinary excretion of l-dopa, and although it did not change urinary dopamine, it increased significantly (P < 0.05) the excretion of the deaminated metabolite of dopamine, DOPAC (Fig. 9). There was no difference in urinary volume, urinary excretion of Na+, norepinephrine excretion, the excretion of the deaminated metabolite of norepinephrine, DHPG, or plasma levels of l-dopa between control and treated animals (Table 4).
This study shows that the uptake of l-dopa into renal tubular cells, and consequently the production of dopamine by these cells, is inhibited by stimulation of β2-adrenergic receptors. Our results also provide evidence of the presence of this regulatory mechanism in the intact animal as well.
Since cell lines do not always maintain the morphological and biochemical characteristics of their parent cells (14), we chose to use freshly isolated proximal tubular cells to optimize the analysis of receptor function. The regulation of the uptake ofl-dopa should be demonstrated at concentrations within the physiological range to be considered crucial in the modulation of renal dopamine synthesis. For this reason, we have studied the transport ofl-dopa into isolated rat renal proximal tubular cells at concentrations lower than those used by other authors (9,25, 26, 28). The rationale for the use of lower concentrations of l-dopa is based on two facts. First, l-dopa concentration in rat and human plasma is about 10 nM (Ref. 17 and present results). Becausel-dopa is reabsorbed from ultrafiltrated plasma, the concentration of the amino acid in the luminal fluid cannot be higher than the plasma concentration. Second, using brush-border membrane vesicles, we have previously studied the uptake of l-dopa by the luminal membrane. In this preparation, the uptake ofl-dopa into vesicles was found to have aK m of about 1 μM (2). In the experimental conditions used in the present study, Scatchard analysis of l-dopa uptake into isolated proximal tubular cells revealed two uptake sites for the amino acid, one with high affinity and low capacity and the other with a lower affinity and a higher capacity. The high-affinity and low-capacity site described here has aK m of the same order of magnitude as the uptake site we have already described using rat renal brush-border membrane vesicles (2). Whether these two sites are identical remains an open question.
Other studies have analyzed the uptake of l-dopa in slices of renal cortex (12), microdissected renal proximal tubules (25), LLC-PK1 cells, a porcine-derived proximal renal tubule epithelial cell line (9,26), and in OK cells, an epithelial cell line derived from the kidney of a female American opossum (28). All these studies were performed in the presence of micromolar to millimolar concentrations of l-dopa and reportedK m values for the transport of the amino acid from 50 to 247 μM and V max values from 0.2 to 5 nmol · mg protein−1 · min−1. The data reported by other authors and the results from the present study suggest that the transport of l-dopa into renal tubular cells involves more than one transport system, as has been described for the tubular transport of other amino acids (18). If this was the case, studies performed at differentl-dopa concentrations could reflect the activity of different transport systems.
The presence in the incubation medium of nonselective adrenergic agonists or a nonselective β-agonist decreased l-dopa uptake by tubular cells. This effect was reversed by the addition to the medium of a β2-antagonist, suggesting that it is mediated by a β2-adrenergic receptor. Moreover, the stimulation of β2-receptors by terbutaline decreasedl-dopa uptake into and the production of dopamine by tubular cells in vitro. The effect on l-dopa uptake was mimicked by the addition of dibutyryl-cAMP, a nonhydrolyzable analog of cAMP, the second messenger triggered upon stimulation of β2-adrenergic receptors.
Structurally similar compounds could potentially compete for a common transporter and result in a reduction in l-dopa uptake. This, however, does not seem to be a likely explanation for the inhibitory effect on l-dopa uptake we are reporting here, since 1) only the nonselective or β2-selective adrenergic agonists, but not the α-selective agonists, elicited an effect; 2) the effect was mimicked by the second messenger cAMP; 3) it was antagonized only by a β2-antagonist; and 4) the time course of the inhibition of the uptake is not compatible with a competitive effect.
β2-Adrenergic receptors have been identified in renal tubular cells (22), but the physiological role of these receptors in proximal tubules is not completely understood. These results provide evidence for a role of β2-receptors in the modulatory mechanisms contributing to the regulation of the intracellular availability of the dopamine precursor. A role of α-adrenergic receptors on uptake of l-dopa was ruled out since neither the inhibitory response to nonselective adrenergic agonists was affected by the presence of selective α-adrenergic antagonists nor the selective α-adrenergic agonists used had any demonstrable effect.
Few studies have described the short-term regulation of amino acid transport in tubular cells. Changes in plasma membrane amino acid transport activity have been suggested to result from translocation of carriers to and from intracellular stores that may represent reserve pools (18). Thus increased amino acid transport activity appeared to result from the recruitment of additional preformed carriers from intracellular pools (8, 16). As demonstrated by the analysis of the saturation curve obtained in the presence of terbutaline, a β2-receptor agonist, the decrease in the uptake of l-dopa was associated with a decrease in the number of high-affinity uptake sites without changes in the affinity constant. It is tempting to speculate that stimulation of β2-adrenergic receptors could trigger the translocation of l-dopa carriers from the plasma membrane to intracellular pools, resulting in both the decreased number of uptake sites but also in the decreased uptake of the amino acid we report here.
In line with the observations in vitro, the administration of the β2-antagonist ICI-118,551 to intact animals resulted in decreased urinary l-dopa and increased excretion of the dopamine metabolite DOPAC without significant changes in plasmal-dopa levels. Since a significant fraction of urinary DOPAC derives from the metabolization of renal dopamine (29) these effects are consistent with increasedl-dopa uptake, and consequently increased dopamine formation, when stimulation of β2-receptors is impaired. An alternative explanation for the increased urinary DOPAC excretion in treated rats would be that ICI-118,551 inhibited catechol-o-methyl transferase activity switching catecholamine metabolism from the methylated to the deaminated metabolites. This hypothesis is unlikely since the excretion of the deaminated metabolite of norepinephrine DHPG was not altered in ICI-118,551-treated rats. Thus our results are consistent with increased l-dopa uptake when stimulation of β2-receptors is impaired, supporting the modulatory role of these receptors on the uptake of l-dopa in vivo.
In conclusion, the present results provide in vitro and in vivo evidence of the modulation by β2-adrenergic receptors of the nonneuronal synthesis of dopamine in proximal tubular cells.
We thank Dr. Selva Cigorraga and Dr. Eliana Pellizari for helpful discussions.
This work was supported by Consejo Nacional de Investigaciones Cientı́ficas y Técnicas (CONICET) Grant PIP 0573 and by Agencia Nacional de Promoción Cientı́fica y Tecnológica PICT 05–00000–0916. M. Barontini, I. Armando, and S. Nowicki are Senior Investigators of CONICET.
Address for reprint requests and other correspondence: I. Armando, Centro de Investigaciones Endocrinologicas, CONICET, Hospital de Niños “R. Gutierrez”, Gallo 1330 (1425), Buenos Aires, Argentina.
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- Copyright © 2000 the American Physiological Society