1 Laboratorio di Genetica Molecolare, 2 Clinica Pediatrica, 3 Divisione di Pneumologia, and 4 Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, 16148 Genoa, Italy
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ABSTRACT |
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We performed Ussing chamber experiments on cultured human bronchial epithelial cells to look for the presence of electrogenic dibasic amino acid transport. Apical but not basolateral L-arginine (10-1,000 µM) increased the short-circuit current. Maximal effect and EC50 were ~3.5 µA/cm2 and 80 µM, respectively, in cells from normal subjects and cystic fibrosis patients. The involvement of nitric oxide was ruled out because a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester) did not decrease the arginine-dependent current. Apical L-lysine, L-alanine, and L-proline, but not aspartic acid, were also effective in increasing the short-circuit current, with EC50 values ranging from 26 to 971 µM. Experiments performed with radiolabeled arginine demonstrated the presence of an Na+-dependent concentrative transporter on the apical membrane of bronchial cells. This transporter could be important in vivo to maintain a low amino acid concentration in the fluid covering the airway surface.
airway epithelium; arginine transport; nitric oxide; pulmonary surfactant; cystic fibrosis
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INTRODUCTION |
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REGULATION OF THE PHYSICAL and chemical properties of
the apical fluid is one of the major functions of airway epithelium. The fluidity and solute composition of this thin layer, which is
essential for mucociliary clearance in the airways, is dependent on the
activity of ion channels and transporters (1). The mechanisms for
Na+ and
Cl transport in epithelial
cells have been intensively studied in the last years due to their
relevance to the pathogenesis of cystic fibrosis (CF) (2). These
studies have led to the identification of some of the ion channels
involved in fluid absorption and secretion, such as the
amiloride-sensitive epithelial Na+
channel (3) and the CF transmembrane conductance regulator (CFTR) (18).
Much less information is available on the transepithelial transport of other solutes such as amino acids. In this regard, the study of dibasic amino acid transport appears particularly interesting because the effective clearance of arginine and lysine on the airway surface seems to be critical for normal respiratory function. In fact, these amino acids inhibit the surface activity of human surfactant lipid extracts, and an abnormally high amino acid concentration has been found in the airways of infants with respiratory distress syndrome (9). Another disease, lysinuric protein intolerance, which is characterized by altered basic amino acid transport and high amino acid concentrations in the airways, is also associated with a high frequency of respiratory distress syndrome (8).
The study of arginine transport is also important because the intracellular levels of arginine are critical for nitric oxide (NO) production by the airway epithelium (7, 14, 15). NO in the airways may be involved in bactericidal activity, the modulation of immune functions, the ciliary beat control, and ion channel regulation (10, 16, 17).
The present study deals with the characterization of arginine transport across polarized monolayers of cultured human bronchial epithelial cells. Our data indicate the presence of an electrogenic amino acid transporter on the apical membrane. This activity could be essential to maintain a low amino acid concentration in the mucociliary fluid.
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METHODS |
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Long-term cell culture. The culture
method for human bronchial epithelial cells has been previously
described (6). Briefly, human bronchi obtained from lung resections
(non-CF subjects) or lung transplants (CF patients) were incubated
overnight with type XIV protease. Epithelial cells collected by
flushing the bronchial lumen were cultured in plastic flasks with
serum-free LHC9-RPMI 1640 medium (6, 13). This medium allowed
expansion of the initial cell number. To obtain differentiated
preparations, cells were plated at high density on permeable supports
(Transwell-COL, Costar). After 24 h, the serum-free medium was replaced
with a mixture of DMEM-Ham's F-12 medium supplemented with 2% serum
(Fetal Clone II, HyClone), insulin, transferrin, ethanolamine,
phosphoethanolamine, hydrocortisone, triiodothyronine, retinoic acid,
and bovine pituitary extract. The cell monolayers received medium on
both sides (1.5 ml on the apical side and 2.5 ml on the basolateral
side). Bronchial monolayers reached a resistance of 2-4
k · cm2 and
an electrical potential difference (PD) in the range of
40 to
50 mV in this medium (6).
Primary culture. Epithelial cells were obtained from a nasal polyp after overnight digestion with protease XIV at 4°C. Epithelial cells were seeded at a density of 3 × 106/cm2 on Snapwell (Costar) permeable supports. As described by Yamaya et al. (23), cells were kept for 24 h in DMEM-Ham's F-12 medium supplemented with 5% fetal calf serum. Subsequently, the basolateral medium was replaced by DMEM-Ham's F-12 medium plus 2% Ultroser G serum substitute (IBF Biotechnics), whereas the apical fluid was removed. Experiments were performed after 6 days.
Ussing chamber experiments. After 5 days in culture, Transwell cups were mounted in a modified Ussing chamber (miniperfusion system Trans-24, World Precision Instruments, Sarasota, FL). Experiments were performed with a Krebs bicarbonate (KB) solution that contained (in mM) 126 NaCl, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. The apical and basolateral solutions were heated at 37°C and continuously bubbled with 5% CO2-95% air. The transepithelial PD was short-circuited with a voltage clamp (558-C5, Department of Bioengineering, The University of Iowa, Iowa City) connected to the apical and basolateral chambers via Ag-AgCl electrodes. The PD and the fluid resistance between potential-sensing electrodes were compensated for. The short-circuit current (Isc) was recorded simultaneously with a chart recorder (Linseis L6512, Selb, Germany) and a MacLab/200 data-recording system connected to a Power Macintosh 4400/200 computer. The final layouts of Figs. 1-9, presenting Ussing chamber experiments, were prepared from computer-stored data with the Igor Pro 3 software package (WaveMetrics, Lake Oswego, OR).
[3H]arginine transport. Both sides of each filter were washed with and equilibrated for 15 min in the KB solution described in Ussing chamber experiments. Subsequently, the apical medium was replaced with 1.5 ml of the prewarmed KB solution containing 100 µM L-arginine and 1 µCi/ml of [3H]arginine. Every 5 min thereafter, for a period of 20 min, aliquots of 20 and 200 µl were removed from the apical and basolateral media. After each sampling, the filter was rapidly moved to another well containing 2.5 ml of fresh saline solution to change the basolateral medium. The entire experiment was performed at 37°C in a humidified atmosphere of 5% CO2-95% air. The radioactivity in the samples taken during the experiment was determined by liquid scintillation counting to calculate the amount of arginine remaining in the apical medium at each time and the efflux of radioactivity at the basolateral side.
In another set of experiments, radioactive arginine was placed at the same concentration (100 µM) on both sides of monolayers. Aliquots of 20 µl were removed every 10 min from each chamber for a period of 1 h to determine the concentration of arginine. Amino acid analysis. Bronchial monolayers were incubated for 1 h with 250 µM arginine in 1.5 ml of KB solution on the apical side. The basolateral side was bathed with 2.5 ml of the same solution without amino acid. At the end of the incubation, apical and basolateral media were removed to determine the amino acid composition with ion-exchange chromatography with a Biochrom 20 (Pharmacia) equipped with a high-resolution column and ninhydrin detection. The results were compared with those obtained from control experiments in which arginine was not included in the apical solution. Materials. The LHC9 medium was prepared from LHC (Biofluids, Rockville, MD) with the addition of supplements as described by Lechner and LaVeck (13). Other culture media and Fetal Clone II were from HyClone (Cramlington, UK). S-nitroso-N-acetylpenicillamine (SNAP) and NG-nitro-L-arginine methyl ester (L-NAME) were from RBI (Natick, MA). All other chemicals were from Sigma (St. Louis, MO). Statistics. The data are presented as representative traces or as arithmetic means ± SE. Unpaired groups of data were compared with Student's t-test to assess significance. ![]() |
RESULTS |
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Transepithelial arginine transport can be electrogenic, as in the
rabbit conjunctiva (12). Therefore, we performed Ussing chamber
experiments on bronchial monolayers with the attempt to show a possible
effect of this amino acid on the
Isc. Basolateral arginine application was uneffective
(n = 3 monolayers; data not shown). On
the contrary, arginine induced a sustained
Isc increase when
added to the apical chamber (Fig.
1A).
The effect of arginine was dose dependent and showed saturation at
concentrations 500 µM. All Ussing chamber data presented were
obtained in the presence of 100 µM amiloride to block the epithelial
Na+ channel. However, apical
L-arginine (100 µM) was also
effective in the absence of amiloride
(n = 3 monolayers; data not shown). The dose-response relationship of the arginine-dependent current could
be fitted with a Michaelis-Menten equation, giving a maximal current and an EC50 around 3.5 µA/cm2 and 80 µM, respectively
(Fig. 1B). The current induced by
arginine was strongly dependent on the active ion transport produced by Na+-K+-ATPase
because it was inhibited by 100 µM ouabain (Fig.
1C).
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NO is produced by enzymatic conversion of arginine to citrulline. NO may have a regulatory role on ion channels such as CFTR (5). Therefore, it was important to assess whether the arginine-dependent current was in part mediated by NO. Various experiments were carried out to elucidate this point. In particular, we asked whether an NO donor could mimic the arginine effect. Accordingly, SNAP (500 µM) was added to the apical and basolateral solutions. This treatment induced a current that was transient and much smaller (<0.2 µA/cm2) than that induced by the subsequent application of arginine (n = 3 monolayers; Fig. 2A). In another set of experiments, we used L-NAME, an inhibitor of NO synthase (NOS). With 500 µM L-NAME on both sides of the epithelium, apical arginine (100 µM) induced an Isc increase of 2.63 ± 0.52 µA/cm2 (n = 3 monolayers; Fig. 2B). This value is comparable with that elicited by arginine in the absence of the inhibitor (2.42 ± 0.32 µA/cm2; n = 8 monolayers).
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Our data indicated that NO synthesis was not involved in arginine-dependent current and favored instead the hypothesis of an electrogenic amino acid transporter. Accordingly, we tested the effect of lysine that, unlike arginine, is not a substrate for inducible NOS. Apical lysine increased the Isc in a way that was very similar to that of arginine (Fig. 3A). Furthermore, arginine was uneffective if applied after a saturating concentration of lysine (Fig. 3B). This result suggested the presence of a common transporter for arginine and lysine.
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The main basic amino acid transport system in various cells is called system y+ (4). This transporter is Na+ independent, inhibited by neutral amino acids, and characterized by transstimulation. There have been fewer descriptions of an Na+-dependent transporter, termed system B0,+, which is also able to transport neutral amino acids (4, 11, 12). To assess the type of transport underlying the current measured in the Ussing chamber experiments, we tested the effect of Na+ removal from the apical solution. Accordingly, L-arginine was applied when the apical solution contained N-methyl-D-glucamine instead of Na+. Under this condition, arginine was largely ineffective compared with the current elicited with apical Na+ on the same epithelium (n = 3 monolayers; Fig. 4A). Similarly, lysine-dependent current was also strongly affected by apical Na+ removal (n = 3 monolayers; Fig. 4B). The Na+ dependence therefore indicated that system B0,+, and not system y+, was the transporter accounting for the effects of arginine and lysine. This conclusion was further supported by the ability of neutral amino acids such as alanine and proline to induce a sustained current (Fig. 5). Note that, in contrast to the other amino acids, the proline-dependent current did not reach saturation at concentrations < 1 mM. Arginine was ineffective if applied after a saturating concentration of alanine or proline (data not shown).
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CFTR appears to have a pleiotropic effect on different transport systems in epithelial cells (19, 22). Therefore, it appeared worthwhile to study bronchial monolayers obtained from CF patients to check whether the response to arginine was altered. As shown in Fig. 6 and Table 1, the behavior of CF cells was similar to that of normal cells in terms of dose-response relationhips.
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A summary of the results obtained with the different amino acids is shown in Table 1. Assuming that EC50 values reflect true Michaelis-Menten constant values, it appears that the transporter has the highest affinity for alanine (25.6 µM) and the lowest for proline (971 µM). Other compounds such as aspartic acid and taurine were ineffective at a concentration of 100 µM (n = 3 monolayers; data not shown). This pattern of amino acid selectivity is consistent with a B0,+ system.
To demonstrate that arginine is taken up at the apical membrane, we placed the bronchial monolayers in open-circuit conditions with [3H]arginine on the apical side. The amount of arginine remaining on this side and the radioactivity released at the basolateral side were determined as described in METHODS. As shown in Fig. 7A, there was a continuous arginine uptake from the apical solution. After 20 min, total arginine transport reached 7.54 ± 0.21 nmol/cm2 (n = 7 monolayers). This corresponds to a concentration decrease in the apical medium from 100 to 76 µM. During the same period, there was a constant release of radioactivity from the basolateral side of the epithelium (Fig. 7B). Assuming that this is due to transepithelial arginine transport, a total basolateral efflux of 1.25 ± 0.02 nmol/cm2 can be calculated during the 20-min period. These results indicate that a significant fraction of arginine removed from the apical solution is accumulated into the cells.
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These experiments were repeated with an Na+-free apical solution. Under this condition, the total arginine uptake in 20 min was significantly reduced to 1.70 ± 0.61 nmol/cm2 (P < 0.001; n = 3 monolayers; Fig. 7A). Basolateral release was also significantly reduced but to a much lesser extent (Fig. 7B).
To strengthen the results obtained with the Ussing chamber experiments, we measured the apical arginine transport in the presence of 100 µM lysine. We reasoned that lysine would have an inhibitory effect if a basic amino acid transporter was present in the apical membrane. We found that [3H]arginine uptake was reduced by >50% in the presence of lysine (P < 0.01; Fig. 7A).
We also wanted to assess the effect of a high concentration of arginine in the basolateral solution. As shown in Fig. 7A, apical arginine uptake was almost unchanged despite 1 mM arginine on the opposite side. Conversely, the basolateral release was significantly increased from 1.25 ± 0.02 to 3.71 ± 0.03 nmol/cm2 (P < 0.001; Fig. 7B).
To further assess the ability of bronchial monolayers to remove amino acids from the extracellular medium, we placed the same concentration of [3H]arginine in both the apical and basolateral solutions. Figure 8 shows that arginine strongly decreased only in the apical side. After 1 h, the concentration changed from 100 to ~50 µM. These experiments demonstrate that bronchial monolayers can actively remove arginine from the apical medium even in the presence of a starting symmetrical distribution.
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Arginine transport was also determined by a nonradioactive method as described in METHODS. This procedure also detected the presence of other amino acids. Consistent with the results of the experiments performed with the radioactive tracer, apical arginine decreased dramatically after 1 h of incubation (from 250 to 121.5 µM), and only a fraction of the arginine removed from the apical medium was released by bronchial cells at the basolateral membrane (Table 2). This analysis also showed a significant efflux of other amino acids, namely, glutamine, glycine, alanine, and threonine. This efflux was not induced by exogenous arginine because it was also observed when the monolayers were kept in the arginine-free saline solution.
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The culture method used for this study differs from more conventional
primary cultures performed in other laboratories. We wondered whether
the expression of the amino acid transporter described here was an
artifact produced by long-term culture of bronchial epithelial cells.
Accordingly, we performed Ussing chamber experiments on nasal
epithelial cells in primary culture. The method used in this case gives
monolayers that resemble the native epithelium (23). Under these
conditions, nasal cells developed a resistance of 850 ± 28 · cm2 and a
transepithelial PD of
57.8 ± 1.8 mV
(n = 4 monolayers). Apical
L-lysine increased the
Isc in a
dose-dependent fashion (Fig. 9). The mean
value of EC50 was 61.2 ± 5.8 µM (n = 4 monolayers), very close to
that found in long-term cultures (Table 1). The maximal current was
significantly larger (12.20 ± 0.85 µA/cm2;
P < 0.01), thus indicating a higher
number of active transporters.
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DISCUSSION |
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Ussing chamber experiments performed on polarized monolayers of human bronchial epithelial cells revealed that apical arginine elicits a transepithelial current. This phenomenon is dose dependent, with an EC50 of ~80 µM. We considered the possibility that the effect of arginine was due to the synthesis of NO by NOS and that NO could, in turn, activate ion channels that would be responsible for the Isc increase. Actually, it has been shown that NO activates the CFTR in T lymphocytes (5). Our results rule out this hypothesis. First, the NO donor SNAP was unable to elicit a sustained Isc increase. Second, L-NAME, an NOS inhibitor, did not decrease the current evoked by arginine. Third, the Isc could also be increased by lysine, alanine, and proline, which are not substrates for NOSs. Saturating concentrations of one amino acid prevented the effect of the other amino acids, thus indicating the presence of a common mechanism.
Ion replacement experiments showed that the current induced by amino acids is strongly dependent on apical Na+. Our data are therefore consistent with the presence of an Na+-dependent electrogenic amino acid transporter on the apical membrane of the epithelium. This transporter can be classified as system B0,+ because it transports basic and neutral amino acids and is Na+ dependent. These characteristics excluded the more widely expressed system y+ arginine transporter, which is defective in lysinuric protein intolerance (21), because it is Na+ independent and inhibited by neutral amino acids.
Experiments performed with radiolabeled arginine confirmed the presence of an amino acid transporter in the apical membrane. Indeed, bronchial monolayers actively removed arginine from the apical but not from the basolateral medium. The inhibition of arginine uptake by lysine further supported the hypothesis of a common transporter for both amino acids.
Apical Na+ removal significantly inhibited arginine uptake. However, the inhibition was not total as that observed for the arginine-dependent current. This could suggest the presence of an additional nonelectrogenic mechanism for arginine transport.
The experiments performed in asymmetric conditions, i.e., with [3H]arginine only on the apical side, revealed that apical uptake is not paralleled by an equal arginine release at the basolateral membrane, at least in open-circuit conditions. This observation was also confirmed by a nonradioactive method. However, the rate of basolateral [3H]arginine efflux was strongly stimulated by arginine application in the basolateral solution. This behavior, termed transtimulation, suggests that the basolateral membrane possesses a system y+-like basic amino acid transporter (4). Actually, this system also has a basolateral localization in other polarized cells (20). The transport across system y+ is controlled by the transmembrane electrical PD so that basic amino acid uptake, and not efflux, is favored by a physiological negative membrane potential. This characteristic could explain the slow basolateral efflux in our experiments because arginine should move along an unfavorable electrical field.
In conclusion, our data indicate that polarized cultures of human
bronchial epithelial cells possess an electrogenic transporter in the
apical membrane that couples the influx of various amino acids with
that of Na+. It is probable that
the transepithelial electrical charge movement, measured during Ussing
chamber experiments, is largely due to Na+ and not to the amino acid
transport because similar maximal current values can be obtained with
amino acids with or without a net electrical charge (see Table 1).
Taking into account an
L-arginine-dependent current of
3 µA/cm2, a corresponding
Na+ flux of 9 × 109
eq/cm2 can be calculated for a
time of 5 min according to the relationship between ion transport
(Ji) and
Isc:
Ji = Isc/zF,
where z is the valence and F is the Faraday
constant. This value can be compared with apical arginine uptake (3.4 × 10
9
mol/cm2) occurring during the
same time interval (Fig. 7). The difference can be
accounted for by the different conditions used (short circuit versus
open circuit) and/or a stoichiometry ratio of
Na+ to amino acid > 1.
Transporter activity would explain a previous observation made in our laboratory (6) that the transepithelial PD of cultured bronchial monolayers is less negative in a physiological saline solution than in the culture medium. Actually, the addition of a saturating concentration of arginine, lysine, alanine, or proline to the KB solution in open-circuit conditions shifted the PD close to values measured in culture (results not shown).
The response to arginine is not altered in CF cells. This result rules out any direct or indirect involvement of CFTR in the arginine-dependent electrogenic effect.
Our findings raise some important questions. Is this transporter expressed in vivo? To the best of our knowledge, there are no reports of a similar transporter in the human airway epithelium. There is only a description of an Na+-dependent lysine transporter in the bullfrog alveolar epithelium (11). The results obtained from the primary culture rule out the possibility that transporter expression is an artifact induced by long-term culture. If this transporter is present in vivo, it would be important to establish whether it is constitutively expressed or subject to modulation and whether it is localized in particular regions of the airways. Of course, a very relevant question regards its physiological role. We can simply speculate that such a transporter, characterized by broad specificity and large transport capacity, could be important to reabsorb amino acids and Na+ from the apical fluid. Such a function could be valuable after a leakage of interstitial fluids due to epithelium damage or at the birth when fluids and solutes filling the airways have to be rapidly reabsorbed.
Some observations suggest an abnormal amino acid concentration in the airway surface fluid of patients with respiratory distress syndrome (8, 9). It would be interesting to evaluate the activity of the apical amino acid transporter in these subjects.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the North American Cystic Fibrosis Foundation (to L. J. V. Galietta).
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FOOTNOTES |
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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: L. J. V. Galietta, Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, 16148 Genoa, Italy.
Received 20 April 1998; accepted in final form 10 July 1998.
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