beta -Alanine and alpha -fluoro-beta -alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2

Mengping Liu1, Rosalind L. Russell1, Leonid Beigelman2, Robert E. Handschumacher1, and Giuseppe Pizzorno1

1 Departments of Internal Medicine (Oncology) and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Ribozyme Pharmaceuticals, Boulder, Colorado 80301


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of beta -alanine as well as alpha -fluoro-beta -alanine (Fbeta AL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for beta -alanine and GABA with Michaelis constant (Km) of 35.3 and 22.5 µM, respectively. A second Na+-dependent kinetic component with Km >1 mM was also identified. The sigmoidal profile of beta -alanine uptake with respect to Na+ shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 ± 0.4 indicates that at least two sodium ions are cotransported with beta -alanine. The flux of beta -alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of beta -alanine to passive diffusion level, indicating that both Na+ and Cl- are essential for the activity of this transporter. The transport of beta -alanine was inhibited by GABA, hypotaurine, beta -aminoisobutyric acid, and Fbeta AL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and alpha -aminoisobutyric acid did not affect beta -alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of beta -alanine and Fbeta AL and their effect on the cellular reuptake of GABA.

uridine; liver; 5-fluorouracil


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

beta -ALANINE AND alpha -fluoro-beta -alanine (Fbeta AL) are the major products of uracil and 5-fluorouracil catabolism. This process occurs primarily in the liver and is specifically localized in parenchymal cells (15). In earlier studies by Diasio and co-workers (25) it was shown that high concentrations of Fbeta AL accumulated initially in kidney and liver, and later on it was found in several other tissues, including eyes and small intestine. These data suggested that an active concentrative process may be responsible. In unrelated studies by Miyamoto et al. (16) and Turner (23), the presence of an active Na+-dependent concentrative mechanism for beta -alanine and beta -amino acids was demonstrated in brush-border vesicles from small intestine and proximal renal tubules (23). Other studies in astrocytes and neurons also established the ability of these cells to concentrate beta -alanine by a Na+-dependent process (9, 14).

Our interest arises from studies on the localization of uridine degradation products in the liver (15). We confirmed that beta -[3H]alanine and [3H]Fbeta AL accumulated in the hepatocytes following incubation with [6-3H]uracil and [5,6-3H]fluorouracil. We also demonstrated these beta -amino acids were concentrated by a Na+-dependent process. Others have characterized the transport of taurine, another beta -amino acid, in rat hepatocytes, showing a single Na+-dependent transport system at low concentration of taurine (5, 8). The transport of taurine was shown to be competitively inhibited by beta -alanine and hypotaurine (8). Two distinct Na+-dependent beta -amino acid transport systems, one with high affinity and the other with low affinity and high capacity, have been characterized in two renal epithelial cell lines, LLC-PK and Madin-Darby canine kidney (MDCK), and in the proximal tubule cells from rabbit and human origin (10-12).

In this study we characterize the Na+- and Cl--dependent beta -alanine and Fbeta AL transporter and provide evidence that it is identical to the GABA transporter designated GAT-2 on the basis of kinetic, substrate, immunological, and inhibition studies.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Chemicals. beta -[3-3H]alanine (87 Ci/mmol) and [3H]H2O (18 Ci/mmol) were purchased from NEN (Boston, MA). [2,3-3H]GABA (91 Ci/mmol) and [carboxy-14C]inulin (10 mCi/mmol) were purchased from Amersham (Arlington Heights, IL). Taurine, hypotaurine, L- and D-alanine, alpha -aminoisobutyric acid, and beta -aminoisobutyric acid were obtained from Sigma Chemical (St. Louis, MO). (R,S)-Fbeta AL was purchased from American Tokyo Kasei (Portland, OR). All other reagents were of the highest grade available.

Hepatocyte preparation. Rat hepatocytes, provided by the generous assistance of the Yale Liver Center, were isolated by the collagenase perfusion technique (2, 21). Male Sprague-Dawley rats (150-200 g) from Charles River Laboratories were anesthetized with 50 mg/kg pentobarbital by intraperitoneal injection. Heparin (500 U/100 g body wt) was injected into the abdominal vena cava, and the livers were perfused through the portal vein with calcium-free perfusion buffer (pH 7.4, 37°C) saturated with a gas mixture of 95% oxygen and 5% carbon dioxide at 38 ml/min for 10 min. Extending the preperfusion period beyond this time period reduced the metabolic and transport activity of both cell populations. The calcium-free perfusate was changed to buffer containing 0.025-0.05% collagenase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 0.5 mM CaCl2. Perfusion was continued for 10 min at 38 ml/min. The liver was removed from the animal and combed into 50 ml of pH 7.4 L-15 medium (GIBCO, Grand Island, NY) saturated with O2. The cell suspension was filtered through 45-µm mesh nylon fabrics (Tetko), and the filtrate was sedimented at 50 g. The hepatocyte pellet was washed three times with cold L-15 medium and resuspended at 4-6 × 106 cells/ml in L-15 medium. Hepatocyte preparations obtained by Percoll centrifugation (21) were >95% pure, and the cell viability, determined by trypan blue exclusion, was in excess of 85% before use and at least 80% at the end of uptake experiments.

Transport studies. Hepatocyte suspensions were centrifuged at 750 g for 5 min in a Sorvall GLC-4 centrifuge. The pellet was washed twice with 150 mM Na+ or 150 mM choline (Na+ free) Hanks' balanced salt medium containing 5.5 mM D-glucose and 4 mM HEPES buffer (pH 7.4). In Cl--free transport experiments, NaCl and KCl were replaced by their corresponding gluconate salts. The pellet was resuspended in the appropriate medium to give a final cell density of 4-6 × 106 cells/ml.

The oil-stop method for nucleoside transport studies previously reported (6) was used to assess transport. Uptake of beta -[3H]alanine and GABA was initiated by mixing 30 µl of cell suspension with 60 µl of radioactive substrate in Hanks' medium in a 1.5 ml Eppendorf microfuge tube. At appropriate time intervals, 60 µl of the mixture were placed in a 400-µl Eppendorf microfuge tube containing 125 µl of oil (16% Fischer 0121 light paraffin oil and 84% Dow Corning 550 silicon fluid; final specific gravity 1.04 g/ml) layered over 30 µl of 15% trichloroacetic acid and centrifuged in a Beckman model B microfuge for 30 s at 10,000 g. The tubes were cut through the oil layer, and radioactivity in each half was assayed in 5 ml of Ecolite(+) after vigorous vortexing. "Time zero" values for uptake, attributed to the extracellular radioactivity trapped in the cell pellet, were determined by spinning 20 µl of hepatocytes through a layer of radioactive substrate (40 µl) placed over the oil in the oil-stop tube. The intracellular volume of hepatocytes was calculated (6) in all experiments by using [3H]H2O to measure the total water space and [14C]inulin for extracellular space. Radioactivity was determined in a Beckman LS 7000 scintillation counter.

In all the kinetic studies an uptake interval of 2-5 min uptake of beta -[3H]alanine and GABA was utilized; however, a 20-min interval was selected for all competition experiments. Paper chromatography with butanol-acetic acid-H2O (25:4:10) was used to demonstrate that radiolabeled alanine and GABA remained intact after exposure to hepatocytes. More than 90% of radioactivity from beta -alanine or GABA incubated in hepatocyte suspensions for 20 min at room temperature migrated to an area identical to that for beta -alanine or GABA.

Uptake determinations were routinely done in triplicate and repeated with two or three different hepatocyte preparations on different days. The results are presented as the means ± SE. Student's t-test for unpaired samples was used to test the significance of difference among means (P < 0.05 was considered significant). Kinetic data were analyzed using the Enzfitter program (R. J. Leatherbarrow, published by Elsevier-Biosoft) for nonlinear least-squares fit of the Michaelis-Menten equation.

Western blot analysis. Rat hepatocytes and MDCK cells, transfected with GAT-2 cDNA (provided by Dr. Michael J. Caplan, Yale University School of Medicine, New Haven, CT), were solubilized in 2× SDS gel loading buffer (100 mM Tris · HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and heated to 100°C for 10 min. The lysate was separated on a discontinuous 7.5% SDS-polyacrylamide electrophoresis system (7, 13, 19). Separated proteins were transferred to polyvinylidene difluoride membranes (Hybond P, Amersham) and blocked with a 5% casein buffer (5% casein, 0.05% Triton X-100, 0.3 M NaCl, 50 mM citric acid, 0.3 M Tris base, pH 7.6). The membranes were incubated overnight at 4°C with polyclonal anti-GABA transporter antibody (R-22) diluted 1:100 in casein buffer (1). The membranes were washed three times (5 min each) with PBS buffer containing 0.05% Tween 20 (PBST) and incubated with 1:2,000 diluted secondary antibody (donkey anti-rabbit horseradish peroxidase conjugated antibody; Amersham) for 1 h at room temperature. The membranes were then washed with PBST (3 × 5 min) and detected with enhanced chemiluminescence reagents (Amersham).


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Studies of the homeostasis and metabolic fate of uridine in the liver (15) indicated that beta -alanine, the primary breakdown product of uracil, remains in the hepatocytes in high concentrations when they are exposed to uracil, a process dependent on sodium (Fig. 1). Biphasic kinetics (Fig. 2) are observed with a high affinity concentrative component [Michaelis constant (Km) 35.3 ± 4.4 µM and a maximal velocity (Vmax) 22.7 ± 2.5 µM/min]. The apparent Km of the concentrative transporter is significantly greater than circulating levels of beta -alanine and thus may be determinant under physiological conditions. A second Na+-dependent kinetic component is also present with an apparent Km of at least 2 mM. The zero transport time course of accumulation of beta -alanine within the cells is also consistent with a very rapid passive diffusion component that leads to equilibration with the medium followed by active concentration, which only occurs in the presence of sodium. When sodium is replaced by either lithium or choline, beta -alanine still rapidly equilibrates with the intracellular water but is not concentrated (Fig. 1). In the presence of Na+, concentrative transport of beta -alanine, after an initial burst, remains linear for >20 min, and >90% of the radioactivity remained associated with beta -alanine.


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Fig. 1.   Time course of beta -[3H]alanine uptake by rat hepatocytes. Cells were suspended in either Na+-containing medium (bullet ) or Na+-free medium [Li+ replacement (black-diamond ) and choline replacement (+)]. Final concentration of beta -alanine during incubation was 25 µM. After appropriate time intervals uptake was terminated by the oil-stop method as described in MATERIALS AND METHODS. Intracellular concentrations were based on intracellular water volume. Values are means ± SE of triplicate estimates.


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Fig. 2.   Substrate dependence of total and Na+-dependent beta -alanine transport in rat hepatocytes. Hepatocytes were incubated in Na+-containing medium and in Na+-free medium with varying substrate concentrations. Transport was measured as described in MATERIALS AND METHODS. The Na+-dependent component of transport was obtained by subtracting, at each substrate concentration, the Na+-free (choline replacement) part from total (inset represents Eadie-Hofstee plot of beta -alanine uptake by hepatocytes at substrate concentrations up to 2 mM). Michaelis constant values for the high affinity process were determined as described.

To determine the stoichiometric relationship between sodium ion entry and the transport of uridine, the dependence of the rate of transport on sodium concentration was determined. A sigmoidal response was obtained (Fig. 3) which is consistent with a cooperative mechanism that requires at least two and probably three sodium ions for each mole of beta -alanine (Hill coefficient 2.6 ± 0.4). These results suggest a mechanism in which the binding of the first sodium ion increases the affinity of the carrier for the second sodium ion and the participation of a single sodium ion is insufficient to permit the active concentration of beta -alanine.


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Fig. 3.   Effect of external Na+ concentrations on Na+-dependent beta -[3H]alanine transport. beta -alanine (25 µM) uptake in hepatocyte was determined in presence of varying Na+ concentrations. Isosmolarity was maintained by replacement of Na+ with choline. Each point is mean of triplicate determinations. Inset presents linear transformation of data as expressed by the Hill equation.

This Na+-dependent transport system was also Cl- dependent. Substituting this anion with gluconate in the transport buffer reduced the rate of uptake and accumulation of beta -alanine to passive diffusion level (Fig. 4). Increasing concentrations of Cl- resulted in a concentrative uptake of beta -alanine that exhibited a hyperbolic dependence, suggesting that one Cl- is associated with the transport of one molecule of beta -alanine (data not shown).


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Fig. 4.   Time course of beta -[3H]alanine uptake by rat hepatocytes in presence and absence of Cl-. Cells were suspended in either Cl--containing medium or Cl--free medium (gluconate replacement). Final concentration of beta -alanine during incubation was 25 µM. After appropriate time intervals uptake was terminated by oil-stop method as described in MATERIALS AND METHODS. Intracellular concentrations were based on intracellular water volume. Values are means of 2 experiments conducted in triplicate.

The Na+- and Cl--dependent transporter is highly substrate specific. L- or D-alanine, taurine, and alpha -aminoisobutyric acid did not inhibit beta -alanine accumulations at concentrations up to 1 mM (Fig. 5). However, GABA and hypotaurine, the sulfonic acid analog of beta -alanine, were potent inhibitors of this process (Fig. 5 and Table 1). Fbeta AL, the primary catabolic product of 5-fluorouracil, also exhibits inhibitory properties on this transport system. All three compounds display competitive inhibition kinetics (data not shown).


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Fig. 5.   Substrate specificity of beta -alanine transport by rat hepatocytes. Hepatocytes were incubated in Na+-containing medium with beta -[3H]alanine (25 µM) for 20 min in absence or presence of beta -alanine structural analogs using range of inhibitor concentrations (0-1 mM). Relative velocities are expressed as percentage of rate of beta -alanine uptake determined in absence of beta -alanine analogs and represent the mean of triplicate determinations. alpha -AIBA, alpha -aminoisobutyric acid, Fbeta AL, alpha -fluoro-beta -alanine; HT, hypotaurine.

                              
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Table 1.   Inhibition of beta -alanine and GABA uptake by structural analogs in rat hepatocytes

Earlier studies using PCR (4) have demonstrated the existence of a specific GABA transporter, termed GAT-2, present in liver, kidney, as well as in brain. Because of its possible identity with the beta -alanine transporter, we have characterized the transport of GABA in hepatocytes and demonstrated an absolute dependence of the concentrative phase on the presence of Na+. A minor degree of concentration may have occurred when Na+ is replaced by Li+ but not choline (Fig. 6). It is apparent in this system as well as with beta -alanine that GABA rapidly enters cells by a Na+-independent system to approximately equilibrate with the medium. As with beta -alanine, the sodium-dependent uptake exhibits a high affinity for GABA (22.5 ± 2.8 µM) and a somewhat lower Vmax (18.5 µM/min) (Fig. 6 and Table 1).


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Fig. 6.   Time course of [3H]GABA uptake by rat hepatocytes. Cells were suspended in l50 mM NaCl, l50 mM LiCl, or l50 mM choline chloride buffer and final concentration of GABA was 25 µM during uptake measurements. After appropriate time intervals uptake was terminated by oil-stop method as previously described. Values are means ± SE of triplicate determinations.

To establish the identity between the beta -alanine Na+- and Cl--dependent transporter in rat hepatocytes and the GAT-2 transporter, we probed protein extracts from hepatocytes with an anti-GABA transporter antibody R-22 (1). As shown in Fig. 7, an 80-kDa protein corresponding to the GAT-2 transporter was present in the hepatocytes extract and in the lysate from MDCK cells expressing GAT-2, used as positive control.


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Fig. 7.   Western blot analysis of GAT-2 antigen in rat hepatocytes and nonparenchymal cells. Rat hepatocyte and MDCK cell proteins were separated on a 7.5% SDS-PAGE gel and analyzed by Western blot as described in MATERIALS AND METHODS. Lanes 1-3, hepatocyte lysates containing 100, 100, and 50 µg of total protein, respectively; lanes 4 and 5, extracts of MCDK cells transfected with GAT-2 cDNA.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The compartmentalization of uridine catabolism in liver resulting in the specific accumulation of beta -alanine and Fbeta AL, in the case of 5-fluorouracil, led to the characterization of this Na+- and Cl--dependent transporter for beta -amino acids and GABA, a system that had poor affinity for taurine. This transporter requires the presence of both specific cation and anion. Without one or the other no significant active transport of beta -alanine and GABA is observed. Thus to demonstrate the cation dependency of this transporter as in Fig. 1, the chloride ion must be present; similarly, as shown in Fig. 4, to prove anion dependency, the transport buffer must contain sodium ion.

The Hill coefficient of 2.6 ± 0.4 for Na+ and one Cl- for beta -alanine is similar to other beta -alanine and beta -amino acid transport systems previously characterized in rat hepatocytes (5, 8), renal brush-border membranes (23), kidney cells derived from the proximal tubules (10, 11), and intestinal brush-border membranes (16). In all these transport systems, however, taurine was also transported and was able to efficiently inhibit the beta -alanine transporter in these tissues. In rat hepatocytes taurine had only a very limited inhibitory effect on beta -alanine uptake (~40%) even at 1 mM.

Kinetic characteristics, substrate specificity, and antibody recognition all suggest that the Na+- and Cl--dependent transporter here described is the GAT-2 transporter. Unlike other GABA transporters previously identified, studies from Borden et al. (4) have indicated that GAT-2 is present not only in the central nervous system but also in other nonneural tissues such as liver, kidney, and retina.

At the neural level GAT-2 is localized in the leptomeninges surrounding the brain, possibly suggesting its role in regulating GABA levels in the cerebrospinal fluid, therefore affecting GABAergic transmission indirectly, or playing a role in the osmoregulation. GAT-2 is absent in neuronal cultures or in neurons in vivo. The basolateral distribution of GAT-2 in MDCK cells (1) and its presence in the leptomeninges also suggest that this transporter is involved in the clearing of GABA from the cerebrospinal fluid in nonsynaptic regions (3).

Neurological toxicity has been reported following administration of 5-fluorouracil (17, 20), particularly for regimens using 24-h infusion of high-dose 5-fluorouracil (2,600 mg/m2) where approximately 6% of the patients developed treatment-related encephalopathy (24). The clinical manifestations include disorientation, confusion, agitation, seizure, stupor, and coma (24). The affinity of beta -alanine and Fbeta AL for the GAT-2 transporter raises the possibility that the neurotoxicity seen with 5-fluorouracil therapy may relate to an effect of Fbeta AL on the reuptake of GABA, an inhibitory neurotransmitter. Administration of Fbeta AL has been shown in cats and dogs to induce a drowsy and stuporous state and struggling that are similar to those produced by oral administration of 5-fluorouracil (18). Histologically two main changes were described: 1) vacuole formation and 2) necrotic lesions. The distribution of these effects was not diffuse but limited to the cerebellar nuclei and white matter, tectum, and tegumentum of the brain stem. The selective localization of the neuropathological changes may be due to the preferential accumulation of Fbeta AL and an inhibitory effect on the function of the GAT-2 transporter.


    ACKNOWLEDGEMENTS

We thank Dr. Chee-Wee Lee (Dept. of Physiology, National University of Singapore, Singapore) for the helpful discussion in the preparation of this manuscript.


    FOOTNOTES

The Madin-Darby canine kidney cells transfected with GAT-2 cDNA and the R-22 anti-GABA transporter antibody were generously provided by Dr. Michael J. Caplan (Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT). The hepatic cells were kindly supplied by the Liver Research Core Center, which was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34989 and the National Institutes of Health Grant R25 (CA-47883-03).

This work was supported by grants from the American Cancer Society (CH67) and National Cancer Institute (CA-08341, CA-45303, and CA-67035).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: G. Pizzorno, Dept. of Internal Medicine (Oncology), Yale Univ. School of Medicine, New Haven, CT 06520.

Received 8 April 1998; accepted in final form 15 September 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(1):G206-G210
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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