Selectivity properties of a Na-dependent amino acid cotransport system in adult alveolar epithelial cells

Xinpo Jiang1, David H. Ingbar2, and Scott M. O'Grady1

Departments of 1 Physiology and 2 Medicine, University of Minnesota, Minneapolis, Minnesota 55455


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the amino acid specificity of a Na-dependent amino acid cotransport system that contributes to transepithelial Na absorption in the apical membrane of cultured adult rat alveolar epithelial cell monolayers. Short-circuit current was increased by basic, uncharged polar, and nonpolar amino acids but not by L-aspartic acid or L-proline. EC50 values for L-lysine and L-histidine were 0.16 and 0.058 mM, respectively. The L-lysine-stimulated short-circuit current was Na dependent, with a concentration causing a half-maximal stimulation by Na of 44.24 mM. L-Serine, L-glutamine, and L-cysteine had EC50 values of 0.095, 0.25, and 0.12 mM, respectively. L-Alanine had the highest affinity, with an EC50 of 0.027 mM. We conclude that monolayer cultures of adult rat alveolar epithelial cells possess a broad-specificity Na-dependent amino acid cotransport system with properties consistent with system B0,+. We suggest that this cotransport system plays a critical role in recycling of constituent amino acids that make up glutathione, thus ensuring efficient replenishment of this important antioxidant within the alveolar fluid.

sodium absorption; ion transport; hyperoxia; antioxidant; system B0,+


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE SODIUM TRANSPORT across the alveolar epithelium plays an important role in the removal of fluid from the alveolar space (6, 15). Experiments using cultured adult rat alveolar epithelial cells have demonstrated that most of the basal electrogenic Na transport across the epithelium is dependent on amiloride-sensitive Na channels located in the apical membrane (4, 9-11, 14-16, 18, 20). In addition, Na-amino acid cotransport has also been shown to provide a pathway for apical Na entry into adult alveolar epithelial cells and contributes significantly to the basal rate of Na transport across the epithelium (9). Previous flux experiments by Brown et al. (1) demonstrated that uptake of the nonmetabolizable amino acid analog alpha -methylaminoisobutyric acid against its concentration gradient is Na dependent in freshly isolated alveolar type II cells and was responsible for 13% of the total Na influx. A Na-dependent L-alanine transporter and a L-glutamine transporter were previously identified in alveolar epithelial cells (2, 7). More recently, Bukowski et al. (2) and Knickelbein et al. (13) described amino acid transport pathways that play an important role in cysteine and cystine uptake from the extracellular space and alveolar fluid in alveolar type II cells. Two Na-dependent (systems ASC and XAG-) and two Na-independent (systems XC and L) amino acid transport systems were identified, but their localization to either the apical or basolateral membrane could not be determined because flux experiments were not performed with cells in monolayer culture.

In a previous study by Jaing et al. (9), Na-amino acid cotransport was shown to be responsible for the majority of the amiloride-insensitive short-circuit current (Isc) in cultured alveolar epithelial cells, accounting for ~30-35% of the basal current. However, the identity of this Na-dependent cotransport system was not determined. The objective of the present study was to examine the amino acid specificity and Na dependence of this Na-amino acid cotransport system in the apical membrane of cultured adult rat alveolar epithelial cell monolayers and use these properties to classify this transport pathway.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Male Sprague-Dawley rats weighing 150-174 g were purchased from Harlan (Indianapolis, IN). Elastase was purchased from Worthington Biochemical (Freehold, NJ). Rat IgG, DNase I, nonessential amino acids, bovine serum albumin (BSA), L-glutamine, HEPES, and trypsin inhibitor were obtained from Sigma (St. Louis, MO). DMEM, Ham's F-12 nutrient mixture (in a 1:1 ratio), and penicillin-streptomycin were purchased from GIBCO BRL (Life Technologies, Grand Island, NY). Nitex mesh (120 and 40 µm) was purchased from Tetko (Elmsford, NY). Tissue culture treated Transwell polycarbonate filters were obtained from Corning Costar (Cambridge, MA). Phosphate-buffered saline was obtained from Celox Laboratories (Oakdale, MN). Amiloride was obtained from Merck Sharp & Dohme Research Laboratories (West Point, PA).

Cell preparation and culture. Alveolar epithelial cells were isolated from adult rat lungs with a modification of the protocol described by Jiang et al. (9). Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium. The lungs were perfused with solution II (in mM: 140 NaCl, 5 KCl, 2.5 NaH2PO4, 1.3 MgSO4, 2.0 CaCl2, 6 glucose, and 10 HEPES). After removal, the lungs were repeatedly lavaged with solution I (in mM: 140 NaCl, 5 KCl, 2.5 NaH2PO4, 6 glucose, and 10 HEPES) and solution II to eliminate macrophages. The lungs were filled with an elastase-containing solution (2.7 U/ml in solution II) and were incubated at 37°C for 30 min in a shaker bath. Elastase was neutralized with a stop solution [2 mM EDTA, 1% BSA, 0.1% soybean trypsin inhibitor, and 0.15 µg/ml of DNase I in a buffered saline solution (in mM: 136 NaCl, 2.2 Na2HPO4, 5.3 KCl, 10 HEPES, and 5.6 glucose)]. Finely minced tissue was filtered through 120- and 40-µm Nitex mesh. The cells were further purified by panning on IgG-coated culture dishes to remove remnant macrophages and suspended directly in serum-free DMEM-Ham's F-12 medium supplemented with 1.25 mg/ml of BSA, 0.1% nonessential amino acids, 2.0 mM glutamine, 100 U/ml of sodium penicillin G, and 100 µg/ml of streptomycin. The cells were then seeded onto Transwell membrane filters (4.52 cm2, 0.4-µm pore size) at a density of 1.5 × 106 cells/cm2 to prepare confluent monolayers. The medium was changed every other day. The resistance of the monolayers was measured with an epithelial volt-ohmmeter (WPI, New Haven, CT). High-resistance monolayers formed on day 4 or 5. All the measurements were performed on day 5, 6, or 7 after isolation.

Ussing chamber measurements. High-resistance monolayers on Transwell inserts were mounted in Ussing chambers and bathed with standard saline solution (in mM: 130 NaCl, 6 KCl, 3 CaCl2, 0.7 MgCl2, 20 NaHCO3, 0.3 NaH2PO4, and 1.3 Na2HPO4, pH 7.4) at 37°C and bubbled with 95% O2-5% CO2 on each side. Monolayer potential difference (luminal side as reference), Isc, and resistance were measured with voltage-clamp circuitry from JWT Engineering (Overland Park, KS). Workbench data acquisition software (Kent Scientific, CT) was used to record the data. To measure apical membrane current, amphotericin B (10 µM) was added to the basolateral solution to eliminate the basolateral membrane as a barrier to ion movement without the loss of intracellular macromolecules. A potassium methyl sulfate-saline solution (in mM: 120 potassium methyl sulfate, 30 mannitol, 3 calcium gluconate, 0.7 MgSO4, 20 KHCO3, 0.3 KH2PO4, and 10 NaCl) was used as the basolateral solution, and a standard saline solution was used to bathe the apical surface of the monolayer. To measure Na+ dependence of L-lysine transport, the monolayers were initially bathed on both sides with N-methyl-D-glucamine (NMDG)-methyl sulfate-saline solution (in mM: 120 NMDG-methyl sulfate, 20 KHCO3, 3 calcium gluconate, 0.7 MgSO4, 0.3 KH2PO4, 1.3 K2HPO4, and 15 mannitol, pH 7.4). A sodium methyl sulfate solution (in mM: 120 sodium methyl sulfate, 20 KHCO3, 3 calcium gluconate, 0.7 MgSO4, 0.3 KH2PO4, 1.3 K2HPO4, and 15 mannitol, pH 7.4) was used to gradually replace the NMDG-methyl sulfate solution to increase Na+ concentration on both sides equally.

Statistics. All data are means ± SE, and n is the number of monolayers studied. The EC50 values for L-amino acids and Na were determined with a four-parameter logistic function to fit the data. The concentrations of each amino acid and for Na at 50% maximal effect were derived from the equation used to fit the concentration-response relationship. Differences between means were analyzed by using either paired or unpaired t-test as appropriate.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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Figure 1 shows the effects of L-lysine on Isc and apical membrane current. After pretreatment of the monolayers with 20 µM apical amiloride (Fig. 1A), subsequent apical addition of 4 mM L-lysine increased either the Isc in intact monolayers (2.15 ± 0.16 µA; n = 20) or the apical membrane current in permeabilized monolayers (3.09 ± 0.40 µA; n = 6). The concentration-response relationships for L-lysine on Isc and apical membrane current were not significantly different (Fig. 1B). The EC50 values for L-lysine on Isc and apical membrane current were 0.16 (n = 5) and 0.18 mM (n = 6), respectively (Fig. 1C). The effect of increasing extracellular Na+ concentration on 4 mM L-lysine uptake was evaluated as shown in Fig. 2. The EC50 for Na dependence of L-lysine transport was 44.24 ± 4.36 mM (n = 4), indicating that a Na-dependent mechanism was involved in L-lysine transport across alveolar epithelial cells. Hill plot analysis of the data revealed a Hill coefficient of 1.41 ± 0.26, suggesting, perhaps, the existence of cooperativity between multiple Na binding sites associated with Na-L-lysine cotransport in alveolar epithelial cells.


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Fig. 1.   A: representative short-circuit current (Isc) trace showing the effect of apical addition of 4 mM L-lysine (n = 12 monolayers). Monolayers were mounted in Ussing chambers and bathed with identical Ringer solution on both apical and basolateral sides. B: representative trace showing effect of apical addition of 4 mM L-lysine on apical membrane current (n = 7 monolayers). Experiments were performed with amphotericin B-permeabilized monolayers mounted in Ussing chambers and bathed with potassium methyl sulfate-saline solution on the basolateral side and standard saline solution on the apical side. C: concentration-response relationships for L-lysine on Isc () and apical membrane current (). The concentrations causing half-maximal stimulation (K0.5) by lysine on Isc and apical membrane current were 0.16 (n = 5 monolayers) and 0.18 mM (n = 6 monolayers), respectively.



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Fig. 2.   Effects of extracellular Na+ concentration on L-lysine transport. Experiments were performed with amphotericin B-permeabilized monolayers mounted in Ussing chambers and bathed on both sides with identical N-methyl-D-glucamine (NMDG)-methyl sulfate-saline solutions. The apical solution contained 4 mM L-lysine. Amiloride (20 µM) was added to the apical solution. A sodium methyl sulfate solution gradually replaced NMDG-methyl sulfate solution to increase Na+ concentration on both sides equally. The K0.5 value for Na dependence of L-lysine transport was 44.24 ± 4.36 mM (n = 4 monolayers).

The effects of acidic (L-aspartic acid), basic (L-lysine and L-histidine), uncharged polar (L-glutamine and L-serine), and nonpolar (L-cysteine, L-alanine, and L-proline) amino acids on Isc across alveolar epithelial monolayers are shown in Fig. 3. After pretreatment of the monolayers with 20 µM apical amiloride, 4 mM L-lysine, L-histidine, L-cysteine, L-alanine, L-glutamine, L-serine, L-proline, or L-aspartic acid was added to the apical solution of the monolayers bathed with identical standard saline solutions on both sides (Fig. 3A). The Isc stimulated by either L-lysine, L-cysteine, or L-glutamine was not significantly different from each other. The Isc stimulated by L-histidine, L-alanine, or L-serine was also not significantly different from each other. However, the L-lysine-, L-cysteine-, or L-glutamine-sensitive currents were significantly greater than the L-histidine-, L-alanine-, or L-serine-activated currents. L-Proline (4 mM) and L-aspartic acid (4 mM) did not produce any response in alveolar epithelial cell monolayers, indicating that Na-dependent transport of acidic amino acids or imino acids could not be detected in these monolayers. From the concentration-response relationships for L-lysine, L-cysteine, L-alanine, L-glutamine, L-serine, and L-histidine on Isc, we found that the rank order of potency was L-alanine > L-histidine > L-serine L-cysteine > L-lysine > L-glutamine (Fig. 3B). To further define the transport pathway for different groups of amino acids, a series of experiments was performed to test for possible additive effects of different classes of amino acids on Isc that might suggest the presence of multiple Na-amino acid cotransporters in the apical membrane (Fig. 4). When 4 mM L-cysteine, L-alanine, L-glutamine, L-serine, or L-histidine was added to the apical surface of the monolayer after 4 mM L-lysine pretreatment, none of these amino acids significantly changed the Isc stimulated by L-lysine. After apical addition of 4 mM L-cysteine, L-alanine, L-glutamine, L-serine, or L-histidine to the monolayers pretreated with 20 µM apical amiloride, 4 mM L-lysine was added to the apical solution. Subsequent addition of L-lysine did not significantly change the Isc stimulated by other amino acids. The results of these experiments indicate the presence of a common Na-dependent amino acid transport system for all of the amino acids studied


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Fig. 3.   Effects of different classes of amino acids on Isc. Monolayers were pretreated with 20 µM apical amiloride and mounted in Ussing chambers. The monolayers were bathed with identical saline solutions on both the apical and basolateral sides. A: amino acid-activated Isc by different classes of amino acids. L-Lysine, L-glutamine, and L-cysteine represent basic, uncharged polar, and nonpolar amino acids, respectively. The Isc values for L-lysine-, L-cysteine-, L-alanine-, L-glutamine-, L-serine- and L-histidine-activated currents were 2.15 ± 0.16 (n = 20 monolayers), 2.19 ± 0.18 (n = 16 monolayers), 1.33 ± 0.15 (n = 21 monolayers), 1.86 ± 0.21 (n = 15 monolayers), 1.25 ± 0.15 (n = 14 monolayers), and 1.31 ± 0.21 (n = 7 monolayers) µA, respectively. B: concentration-response relationships for L-lysine, L-cysteine, L-alanine, L-glutamine, L-serine, and L-histidine on Isc. L-Histidine, L-alanine, and L-serine represent basic, nonpolar, and uncharged polar amino acids. The K0.5 values for L-lysine, L-cysteine, L-alanine, L-glutamine, L-serine, and L-histidine on Isc were 0.18 (n = 5 monolayers), 0.12 (n = 5 monolayers), 0.027 (n = 6 monolayers), 0.25 (n = 5 monolayers), 0.095 (n = 6 monolayers), and 0.058 (n = 5 monolayers) mM, respectively.



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Fig. 4.   A: nonadditive effects of L-cysteine (C), L-alanine (A), L-glutamine (Q), L-serine (S), and L-histidine (H) on L-lysine (K) transport. Monolayers pretreated with 20 µM apical amiloride were mounted in Ussing chambers and bathed with identical saline solutions on both the apical and basolateral sides. After apical addition of 4 mM L-cysteine (n = 4 monolayers), L-alanine (n = 5 monolayers), L-glutamine (n = 5 monolayers), L-serine (n = 4 monolayers), or L-histidine (n = 7 monolayers), 4 mM L-lysine was added to the apical solution. B: nonadditive effects of L-lysine on transport of different classes of amino acids. Monolayers were mounted in Ussing chambers and bathed with identical saline solutions on both the apical and basolateral sides. After apical addition of 4 mM L-lysine, 4 mM L-cysteine (n = 4 monolayers), L-alanine (n = 4 monolayers), L-glutamine (n = 4 monolayers), L-serine (n = 4 monolayers), or L-histidine (n = 4 monolayers) was added to the apical solution.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Amino acid transport systems are widely distributed among different cell types and have broad specificity for neural, basic, acidic, imino, and aromatic amino acids. Based on functional studies of saturation of transport, substrate specificity, kinetic behavior, and mechanisms of regulation performed in perfused organs, isolated cells, and purified plasma membranes, amino acid transport systems have been divided into three groups: zwitterionic amino acid transporters, cationic amino acid transporters, and anionic amino acid transporters (17). Each category can be divided into Na+-dependent and Na+-independent subgroups. The Na+-dependent amino acid transport systems represent a particularly important class in epithelial tissues because they facilitate sodium uptake and may play an important role in transepithelial Na absorption. System A, system ASC, the imino acid transport system, system B0 (zwitterionic amino acids), system B0,+ (cationic amino acids), and system XAG- (anionic amino acids) are Na+-dependent transporters expressed in epithelia. System A is located in the basolateral membranes in the intestine (19) and has been identified in alveolar epithelial cells (5). Systems B0 and B0,+ are broad specificity amino acid transporters that are also commonly found in epithelial cells (17). System XAG- has been identified in alveolar epithelial cells and has been shown to transport L-cysteine and L-cystine in addition to L-aspartic acid and L-glutamine (7, 13).

In this study, we examined the amino acid specificity of a Na-dependent amino acid cotransporter present in the apical membrane of adult rat alveolar epithelial cells that was previously shown (9) to contribute to transepithelial Na absorption. Our results indicate that this cotransporter has broad specificity for different classes of amino acids. We observed that the EC50 values for L-lysine on the Isc in alveolar epithelial monolayers and on the apical membrane current in permeabilized monolayers were nearly identical. Because permeabilized monolayers were bathed with identical solutions on both the apical and basolateral sides (except for 4 mM L-lysine in the apical solution), the L-lysine-dependent current that increased as a function of extracellular Na+ concentration was produced by Na-dependent amino acid cotransport activity driven by the L-lysine gradient. Isc was stimulated by basic, uncharged polar, and nonpolar amino acids but not by an acidic amino acid or proline, suggesting the presence of Na-dependent system B0,+ in alveolar epithelial cell monolayers. This cotransport system transports zwitterionic and basic amino acids but not L-proline or L-aspartic acid (17).

In the present study, amino acids with low affinity for the cotransporter tend to produce more current than those with high affinity for the cotransporter. This difference may be due to a slower turnover rate of the cotransporter when amino acids with high affinity are bound, which results in a decreased rate of debinding from the cotransporter once the amino acid enters the cell. Apical addition of L-lysine to the monolayers pretreated with any amino acid with high affinity for the cotransporter did not significantly change the Isc. In fact, the effects of the various classes of amino acids on Isc were nonadditive no matter which amino acid was added first to the apical solution, indicating that all the amino acids tested were transported by the same Na-dependent amino acid cotransporter, most likely system B0,+. It is worth noting that the approach used in this study to examine Na-amino acid cotransport gave a concentration causing half-maximal stimulation (K0.5) for L-alanine transport that was consistent with that reported by Clerici et al. (5). In this earlier study, tracer flux measurements were used to determine the kinetics of L-alanine transport in rat alveolar type II cells. In contrast, the K0.5 value for L-cysteine was more than sevenfold greater than that previously reported for system ASC present in alveolar type II cells but similar to that reported for system XAG- (13).

Measurements of Isc in cultured adult rat alveolar cell monolayers indicate that Na-dependent amino acid cotransport provides an additional pathway for Na uptake across the apical membrane. The physiological significance of this transport mechanism is not completely clear, but it is possible that it functions in the recovery of free amino acids that leak into the alveolar space from the plasma. It may also serve to recover amino acids produced as degradation products of proteins or peptides that are released into the alveolar space. The observation that this Na-dependent amino acid cotransporter transports L-cysteine suggests that it contributes to the production of glutathione within alveolar epithelial cells (13). Glutathione is an important antioxidant that protects cells from reactive oxygen metabolite-induced injury that can occur under both normoxic and hyperoxic conditions. It is present in relatively high concentrations within the alveolar fluid (3). L-Cysteine availability is considered to be a rate-limiting step in glutathione synthesis. Uptake of L-cysteine across the apical membrane may be facilitated by the presence of gamma -glutamyl transpeptidase, an ectoenzyme that breaks down glutathione to generate glutamate and cysteinylglycine (8, 12).

In conclusion, our results suggest that cultured adult rat alveolar epithelial cells possess a Na-dependent amino acid transporter that specifically transports basic, uncharged polar, and nonpolar amino acids. The amino acid selectivity of this cotransporter is consistent with system B0,+, which has not been previously identified in alveolar epithelial cells. We propose that this transporter contributes to electrogenic transepithelial Na absorption under conditions where amino acids transported by this system are present within alveolar fluid. We also suggest that this cotransport pathway plays a role in the recovery of L-cysteine from the alveolar fluid produced by glutathione degradation that occurs at the apical surface of the alveolar epithelium. Recycling of constituent amino acids that make up glutathione is likely to ensure efficient replenishment of this important antioxidant compound within the alveolar fluid.


    ACKNOWLEDGEMENTS

We thank Peter Maniak for technical assistance on this project.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Specialized Center for Research Grant HL-50152.

Address for reprint requests and other correspondence: S. M. O'Grady, Dept. of Physiology and Animal Science, Univ. of Minnesota, 495 Animal Science/Veterinary Medicine Bldg., 1988 Fitch Ave., St. Paul, MN 55108 (E-mail: ograd001{at}tc.umn.edu).

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. Section 1734 solely to indicate this fact.

Received 7 January 2000; accepted in final form 30 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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