The regulatory actions of adenosine on ion channel function are mediated by four distinct membrane receptors. The concentration of adenosine in the vicinity of these receptors is controlled, in part, by inwardly directed nucleoside transport. The purpose of this study was to characterize the effects of adenosine on ion channels in A549 cells and the role of nucleoside transporters in this regulation. Ion replacement and pharmacological studies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K+ channels, most likely Ca2+-dependent intermediate-conductance K+ (I K) channels. A1 but not A2 receptor antagonists blocked the effects of adenosine. RT-PCR studies showed that A549 cells expressed mRNA for I K-1 channels as well as A1, A2A, and A2B but not A3 receptors. Similarly, mRNA for equilibrative (hENT1 and hENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleoside transporters was detected, a result confirmed in functional uptake studies. These studies showed that adenosine controls the function of K+ channels in A549 cells and that hENTs play a crucial role in this process.
- A549 cells
- human concentrative nucleoside transporter
- human equilibrative nucleoside transporter
- calcium-dependent intermediate-conductance potassium channels
the purine nucleoside adenosine is an important mediator of many physiological functions, including transepithelial electrolyte secretion in human airways. Adenosine mediates its effects through the activation of high-affinity receptors, A1 and A2A, that are physiologically relevant and low-affinity receptors, A2Band A3, that could play a crucial role under inflammatory conditions (34). Stimulation of adenosine receptors activates anion conduction via both cystic fibrosis transmembrane conductance regulator (CFTR)-dependent and -independent pathways (11, 26, 39), although the underlying mechanisms are not fully understood.
The magnitude of the effect of adenosine on ion transport is related to its concentration in the vicinity of its cell surface receptors. In healthy subjects the average adenosine concentration in airway surface liquid is ∼60 μM, whereas in patients with asthma it is ∼200 μM (14). The increased adenosine concentration in asthmatic patients raises two important questions: What is the source of adenosine and how are its levels controlled? One factor that is believed to control adenosine concentrations is the balance between the activities of enzymes that catalyze its synthesis and those that catalyze its metabolism. Adenosine is produced by the action of membrane-bound 5′-nucleotidase on extracellular AMP, which is itself produced by the action of nonspecific (alkaline or acidic) phosphatases on ADP and ATP (35). Conversely, the metabolism of adenosine is mediated by the action of either adenosine kinase or adenosine deaminase, resulting in the conversion of adenosine to AMP or inosine, respectively (35). Because the majority of adenosine synthesis occurs extracellularly whereas most of its metabolism occurs intracellularly, inwardly directed transport of adenosine across the plasma membrane is also an important determinant of its extracellular concentration (8).
Because adenosine and other nucleosides are relatively hydrophilic, their uptake and release from cells depend on specialized nucleoside transporter proteins present in the plasma membrane (8). These are members of the concentrative (Na+ dependent) nucleoside transporter (CNT) and equilibrative (Na+independent) nucleoside transporter (ENT) families (1, 7,46). Molecular cloning studies in humans and rodents have identified three distinct members of the concentrative family (CNT1, CNT2, and CNT3) and two members of the equilibrative family (ENT1 and ENT2). Human (h)CNT1 and hCNT2 both transport uridine and certain uridine analogs but are otherwise selective for either pyrimidine (hCNT1) or purine (hCNT2) nucleosides, except for modest transport of adenosine by hCNT1 (37, 38, 41). In contrast, hCNT3 transports both purine and pyrimidine nucleosides (36). hENT1 and hENT2 also transport both purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nitrobenzylthioinosine (NBTI), hENT2 being NBTI insensitive (12, 18, 19). They also differ in sensitivity to inhibition by the coronary vasodilators dipyridamole, dilazep, and draflazine (hENT1 > hENT2) and in the ability of hENT2 to transport nucleobases as well as nucleosides.
The concentrative (inwardly directed) nucleoside transporters of rodents and humans are expressed in specialized cells such as intestinal and renal epithelia, liver, choroid plexus, splenocytes, macrophages, and leukemic cells (1, 7, 46). The equilibrative (bidirectional) nucleoside transporters have generally lower substrate affinities than the concentrative transporters and occur in most, possibly all, human and rodent cell types. Although the role of nucleoside transport in the control of transepithelial anion secretion is unknown, it has been recently suggested that CNT2 could play an important role in the regulation of Na+reabsorption in cultured rat epididymal epithelium (28).
The aim of the present study was to characterize the role of adenosine receptors and transporters in the regulation of ion transport in human airway epithelial cell line A549. These cells exhibit metabolic and transport properties consistent with type II pneumocytes and, because they do not express CFTR (16), constitute a convenient model for studying CFTR-independent regulation of ion transport by adenosine. Our data show that regulation of adenosine receptor function by hENTs controls ion transport in A549 cells.
MATERIALS AND METHODS
A549 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 μg/ml gentamicin sulfate, 60 μg/ml penicillin G, and 100 μg/ml streptomycin. Cells were maintained in T25 tissue-culture flasks (Costar, Cambridge, MA) at 37°C in a humidified atmosphere of 5% CO2 in air. Confluent cell layers were passaged using saline solution containing 0.05% trypsin and 0.02% EDTA. For adenosine transport experiments, cells were seeded at a density of 106 cells/cm2onto Costar Snapwell inserts (0.45-μm pore size, 1-cm2surface area) coated with type VI human placental collagen (Sigma, St. Louis, MO). For the first 6 days, cells were grown submerged in culture medium that was changed every 2–3 days. Subsequently, air interface culturing was used, in which the medium was added only to the basolateral side of the inserts. For whole cell patch-clamp studies, cells were seeded onto 35-mm plates (Becton Dickinson, Franklin Lakes, NJ) at a density of 1.5 × 103 cells/cm2at least 4 h before experiments.
Whole cell patch-clamp recordings.
Pipette electrodes were made from thin-walled borosilicate glass (A-M Systems, Everett, WA) with a two-stage vertical puller (Nirashige). Electrode tips were fire polished to a final resistance of 3–6 MΩ immediately before experiments. The composition of pipette and bath solutions is given in Table 1. Cultured cells were rinsed three times in bath solution immediately before being mounted into a holder fixed to the stage of an Olympus IMT-2 inverted research microscope (Lake Success, NY). The holder maintained the bath solution at 37°C by means of a heat-exchange perfusion system. After the pipette had been immersed in the bath solution, offset potentials were compensated before a gigaohm seal was formed. Once sealed, the whole cell configuration was obtained mechanically, by suction, and the cell was immediately clamped to −40 mV. Currents were recorded at 1-min intervals with an Axopatch 200A amplifier and Clampex 8.0 software, both from Axon Instruments (Foster City, CA), in response to the voltage protocol shown in Fig.1 C. All currents were reported with reference to the ground electrode in the bath. The access resistance of the patch and cell capacitance were measured directly by the compensation circuitry of the patch-clamp amplifier and by Clampex 8.0 software. The whole cell capacitance in these experiments, expressed as mean ± SD, was 27 ± 6 pF (n = 125), and only seals with a series resistance of <20 MΩ were used. All data were analyzed by Clampfit 8.0 (Axon Instruments), Microsoft Excel 97 (Seattle, WA), and Micrococal Origin 5.0 (Northampton, MA) software. Traces were first normalized to 1 pF to remove variability due to cell size. The current-voltage relationship was obtained from the mean current during the central 140 ms of the recording. The whole cell current chord conductance (γ) was computed from the equation I = γ(V − E rev), whereI is the whole cell current, V is the applied voltage, and Erev is the whole cell current reversal potential. Calculations of chord conductance were performed atV = 40 mV.
Data are presented as means ± SE; n refers to the number of experiments. The paired Student's t-test was used to compare the means of two groups. Statistically significant differences among the means of multiple groups were determined by one-way analysis of variance (ANOVA) with the Tukey-Kramer post test with the use of GraphPad Instat 3.05 software (San Diego, CA). A value of P <0.05 was considered statistically significant.
Total RNA was isolated from 2 × 106 cells using the Qiagen RNeasy kit. The average amount of RNA obtained from 2 × 106 cells was ∼400 ng. One-fourth of the RNA was reverse transcribed with the use of Superscript II reverse transcriptase (GIBCO BRL) and either oligo(dT) or random hexamers (50 A260units; Boehringer Mannheim) as primers. Thereafter, PCR was performed in 20-μl reactions with the primer pairs (25 μM) described in Table2. In addition to the primers designed to amplify sequences of interest, reactions with glyceraldehyde-3-phosphate dehydrogenase (GAPDH)- specific primers were run in all rounds of PCR reactions to serve as internal positive controls. One-tenth of the cDNA was used in PCR experiments, and amplification proceeded by annealing for 30 s at the temperatures indicated in Table 2, followed by an elongation step at 72°C for 1 min. Sequences were amplified over 30 or 38 cycles, and PCR products with the expected sizes, shown in Table 2, were resolved on 1.5% agarose gels. All RT-PCR products were sequenced in one [adenosine receptors and Ca2+-dependent intermediate-conductance K+ (I K)-1 channels] or both (nucleoside transporters) directions by Taqdideoxyterminator cycle sequencing with an automated DNA sequencer (model 373A, Applied Biosystems, Foster City, CA).
Experiments were carried out at 20°C in HEPES-buffered Ringer's solution (HPBR) containing (in mM) 135 NaCl, 5.0 KCl, 3.33 NaH2PO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, and 5.0 HEPES (pH = 7.4 at 20°C) or in Na+-free HPBR containing (in mM) 140N-methyl-d-glucamine, 5.0 KH2PO4, 1.0 CaCl2, 1.0 MgCl2, 10 glucose, and 5.0 HEPES (pH = 7.4 at 20°C). Confluent monolayers of A549 cells grown on permeable filters were washed six times with HPBR or Na+-free HPBR and then incubated in the same solution (±1 μM NBTI) for 30 min. Uptake was initiated by adding 10 μM 14C-labeled adenosine or uridine (0.5 μCi/ml, Amersham Pharmacia Biotech) in HPBR or Na+-free HPBR (±1 μM NBTI) to either the apical or the basolateral compartment. Incubations with adenosine also included 1 μM deoxycoformycin to inhibit adenosine deaminase activity. Uptake was terminated after 30 s to 3 min by 10 rapid washes of the cell culture inserts in an ice-cold “stop” solution containing (in mM) 100 MgCl2 and 10 Tris · HCl (pH = 7.4 at 0°C) (32). The monolayers were dissolved in 0.2 ml 5% (wt/vol) SDS and counted for radioactivity using a Beckman LS 6000IC liquid scintillation counter (Irvine, CA). Nonmediated (passive) uptake was determined in the presence of 1 μM NBTI and excess (5 mM) unlabeled uridine. The protein content of representative monolayers was measured using the Bio-Rad protein standard assay procedure. The flux values shown are means ± SE of n = 5 inserts. Each experiment was repeated at least three times on different batches of cells.
Adenosine and deoxycoformycin were prepared in H2O as 10 mM and 1 mM stock solutions, respectively. NBTI was prepared as a 3 mM stock solution in methanol, clotrimazole as a 30 mM stock solution in ethanol, and amiloride as a 10 mM stock solution in H2O. All the above drugs were obtained from Sigma. 9-Chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS-15943) was prepared as a 2 mM stock solution in DMSO, 1–3-dipropyl-8-cyclopentylxanthine (DPCPX) was prepared as a 10 μM stock solution in 0.1 N NaOH, and 3,7-dimethyl-1-propargylxanthine (DMPX) was prepared as a 5 mM stock solution in H2O; all three were purchased from RBI (Natick, MA). Finally, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS; Molecular Probes, Eugene, OR) was prepared as a 5 mM stock solution in H2O, 1-ethyl-2-benzimidazalinone (1-EBIO; Aldrich, Milwaukee, WI) as a 600 mM stock solution in ethanol, and 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991, a generous gift from Dr. B. S. Brown, DuPont, Wilmington, DE) as a 10 mM stock solution in 0.1 N HCl.
Effect of exogenous and autocrine adenosine on whole cell current in A549 cells.
Figure 1 A shows typical recordings of whole cell current in A549 cells obtained in nonsymmetrical cationic solutions (pipette 135 mM KCl, bath 135 mM NaCl). Addition of adenosine (100 μM) to the bath solution significantly activated whole cell current (n = 8,P < 0.01). The effect of autocrine adenosine on whole cell current was studied with NBTI, an inhibitor of ENTs. We reasoned that if the uptake of extracellularly produced adenosine were inhibited, its concentration would increase, leading to activation of adenosine receptors. Figure 1 B shows that the addition of NBTI (10 μM) to the bath solution had an effect on the whole cell current that was similar to that caused by the addition of exogenous adenosine (n = 15, P < 0.01). Similar experiments performed with NBTI at a concentration that specifically inhibits hENT1 but not hENT2 (1 μM) showed that there was no significant difference (P > 0.05, n = 6) in whole cell current activation by these two concentrations of inhibitor (data not shown). These results suggested that hENT1 may mediate the majority of nucleoside transport in A549 cells.
The observation that NBTI had no effect on whole cell current in the presence of exogenous adenosine suggests a common mechanism of current activation by action on adenosine receptors (Fig.2). This conclusion was further supported by experiments showing that addition of the nonselective adenosine receptor antagonist CGS-15943 (1 μM) reversed activation of the whole cell current by NBTI (Fig. 3). Interestingly, CGS-15943 alone had no effect on the basal whole cell current (P > 0.05, n = 6; data not shown).
Figure 4 shows the effects of adenosine receptor antagonists on the whole cell current. DPCPX, a specific antagonist of A1 receptors, reversed the effect of adenosine, indicating that these receptors are involved in current activation. In contrast, A2A and A2B receptors appeared not to be involved in this process, because their antagonist, DMPX, had no effect on the current activated by adenosine (Fig. 4,B and D).
Identification of channel types activated by adenosine.
To determine the channel types involved in the response to adenosine, ion replacement and pharmacological studies were performed. Replacement of K+ in the pipette and bath solutions by Cs+reduced basal conductance by 60%, indicating that K+channels contribute to basal whole cell current (n = 3,P < 0.05). Under these conditions, addition of adenosine to the bath solution had no effect on the whole cell current, indicating that K+ channels are the major targets for adenosine action (n = 3, P > 0.05).
Epithelial cells possess two distinct classes of K+channels regulated by cAMP- and Ca2+-mediated agonists, respectively. Therefore, we used selective modulators of these channel families to characterize their contribution to baseline and adenosine-stimulated current. A specific inhibitor of cAMP-dependent K+ channels, XE-991 (10 μM), appeared to affect neither basal whole cell current nor the subsequent response to 100 μM adenosine, suggesting that these channels do not contribute to the cell membrane conductance (n = 4, P > 0.05, paired Student's t-test). In contrast, an opener ofI K channels, 1-EBIO (600 μM), increased the mean cell membrane conductance from 75 ± 5 to 468 ± 35 pS/pF and caused a shift in the reversal potential from −14 ± 3 to −68 ± 2 mV, indicating that these channels make a major contribution to the membrane conductance (n = 4,P < 0.0001). This result was further supported by the use of clotrimazole, a selective inhibitor of I Kchannels (Fig. 5). Clotrimazole (10 μM) inhibited basal whole cell current (P < 0.05,n = 8) and abolished the subsequent response to adenosine, indicating that I K channels were a likely target for adenosine action.
The effect of adenosine on the activity of Na+ and Cl− channels was evaluated using amiloride and DIDS, respectively. Amiloride (10 μM), a specific blocker of epithelial Na+ channels, appeared to affect neither basal whole cell current nor the subsequent response to 100 μM adenosine (Fig.6). In contrast, addition of 50 μM DIDS to the bath solution reduced the whole cell current, indicating that DIDS-sensitive Cl− channels make a contribution to the basal current (Fig. 7). However, adenosine in the presence of DIDS significantly increased the current, indicating that other channels were activated by adenosine. In summary, these results have demonstrated that stimulation of A1receptors in A549 cells activates K+ transport, likely through I K channels.
Identification of adenosine receptors, nucleoside transporters, and IK channels using RT-PCR.
The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors distinguished as A1, A2A, A2B, and A3. Gene expression of these receptors was investigated with RT-PCR. As shown in Fig.8 A, A549 cells express mRNA for A1, A2A, and A2B but not A3 receptors. Interestingly, studies with selective adenosine receptor antagonists indicate that only the A1receptor is involved in the regulation of whole cell current.
Electrophysiological studies indicated that I Kchannels function in A549 cells and are involved in the response to adenosine. Our RT-PCR data confirm the presence of mRNA for theI K-1 protein in A549 cells (Fig. 8 B).
Figure 8 C shows RT-PCR amplification of nucleoside transporter transcripts in A549 cells. The cells contained mRNA for both ENTs (hENT1 and hENT2) but lacked transcripts for CNTs (hCNT1, hCNT2, and hCNT3).
All PCR products were sequenced and found to be identical to corresponding GenBank sequences (accession numbers given in Table 2). Control amplifications in the absence of added A549 cDNA were negative for all successfully identified adenosine receptors (A1, A2A, and A2B), I Kchannels (I K-1), and nucleoside transporters (hENT1 and hENT2), whereas control amplifications done with GAPDH primers were always positive.
Functional studies of nucleoside transport by A549 cells.
Apical and basolateral uptake of adenosine was measured as a function of time at room temperature and, as shown in Fig.9, was linear for 3 min (apical > basolateral). Subsequent initial rate measurements to determine the basolateral and apical pathways for adenosine and uridine transport were carried out using a 2-min incubation and are shown in Fig. 10. Apical transport of adenosine was not significantly reduced by removal of extracellular Na+ but was substantially inhibited by 1 μM NBTI, a concentration sufficient to block all hENT1-mediated transport activity (Fig. 10 A). NBTI-insensitive adenosine uptake was reduced further in the presence of excess unlabeled uridine. Because adenosine and uridine are both transported by hENT1 and hENT2, this result identifies additional hENT2-mediated and passive components of adenosine uptake. At the concentration of adenosine tested (10 μM), the ratio of the contribution of hENT1 (NBTI sensitive) to that of hENT2 (NBTI insensitive), corrected for passive uptake, was 9:1. Basolateral adenosine transport (Fig. 10 B) as well as apical and basolateral transport of uridine (Fig. 10, C andD), a universal ENT/CNT permeant, showed similar characteristics (hENT1-to-hENT2 flux ratios 12:1, 5:1, and 4:1, respectively). These results were consistent with the electrophysiological data, which indicated that the majority of nucleoside transport that effects whole cell current is mediated by hENT1.
Extracellular adenosine was shown previously to modulate the function of both cation (3, 6, 29, 39) and anion channels (5, 6, 10, 26, 33, 39) in several epithelia. There are also reports suggesting that the presence of nucleoside transporters in epithelial cells may regulate these effects by controlling the effective concentration of adenosine in the vicinity of its receptors (28, 32). The results presented in this paper confirm and extend these observations by identifying and characterizing the ion channels, adenosine receptors, and nucleoside transporters involved in the regulation of whole cell current in A549 cells.
In the lung, most of the extracellular adenosine is derived from cleavage of the nucleotide AMP by the enzyme 5′-nucleotidase, which is located on the outer surface of the cell plasma membrane (35). AMP, in turn, may be generated from epithelium-derived extracellular ATP and other adenosine nucleotides, including cAMP. Because ATP is present at millimolar concentrations in the cytoplasm, it is possible that release of ATP from injured airway cells contributes to the increased concentration of adenosine found in the airway surface liquid of asthmatic patients. In addition, it has been suggested that cells may secrete ATP, by Ca2+-dependent vesicular exocytosis or through ATP transporters whose identity is still controversial (22). Intracellular levels of adenosine are normally kept low mainly by its conversion to AMP by the enzyme adenosine kinase, which creates an inwardly directed gradient for adenosine entry into the cell. However, the possibility of direct adenosine release from the airway epithelium cannot be excluded, particularly under conditions of stress.
The results presented in this study show that the application of NBTI, a selective inhibitor of hENT1-mediated adenosine transport, had effects on whole cell current similar to those of the application of exogenous adenosine. Furthermore, the effect of NBTI was not additive with that of adenosine and was inhibitable by the adenosine receptor antagonist CGS-15943, indicating that the effect of NBTI is mediated through the activation of adenosine receptors. Therefore, adenosine transporters could regulate epithelial electrolyte secretion by controlling adenosine concentration in the vicinity of its receptors.
It is important to note that CGS-15943 alone had no effect on the baseline current, indicating that endogenous adenosine does not affect the baseline whole cell current. However, several observations suggest that the effect of autocrine adenosine in vivo may be different from that in vitro. First, epithelial cells are normally covered by a thin (∼10 μm) layer of airway surface liquid in vivo, whereas cells in our experiments were covered by a ∼1-cm-thick layer of bath solution. Second, in vivo, the whole epithelial monolayer contributes to adenosine generation, in contrast to a single cell in patch-clamp studies. Third, other cell types (e.g., mast cells) in the vicinity of the epithelium may contribute to extracellular adenosine concentration in vivo.
The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors, distinguished as A1, A2A, A2B, and A3 (17,25). Activation of each of these receptors has been linked with the regulation of ion transport in epithelial tissues (3, 4, 26,39). The results of RT-PCR experiments have shown that A549 cells express A1, A2A, and A2B but not A3 receptors. However, functional studies with specific adenosine receptor antagonists indicate that only the A1receptor is involved in the regulation of whole cell current by adenosine. Because A1 receptors are linked to Gi1/2/3 proteins and their activation increases inositol 1,4,5-trisphosphate generation and intracellular Ca2+concentration (17), it is likely that adenosine stimulates whole cell current by activation of Ca2+-dependent ion channels.
Adenosine receptor activation has been shown to activate CFTR Cl− channels (11), non-CFTR Cl−channels (5, 33), amiloride-sensitive Na+channels (3, 29), and Ca2+-dependent K+ channels (39). Because A549 cells do not express CFTR (16), they constitute a convenient model for the study of the regulation of non-CFTR anion channels. DIDS reduced whole cell current in A549 cells, indicating significant contribution of non-CFTR anion channels to the basal whole cell current. Interestingly, subsequent application of adenosine activated whole cell current, indicating that DIDS-sensitive anion channels may not be targeted by adenosine.
A549 cells were recently shown to contain amiloride-sensitive Na+ channels with molecular and biophysical properties similar to those of alveolar type II cells (27). However, the data from the present study showed whole cell current activation in the presence of amiloride, suggesting that amiloride-sensitive Na+ channels are not affected by adenosine. Similar to other examples of tissue-specific regulation of Na+channels (for review, see Ref. 31), this result is clearly different from the effect of adenosine on amiloride-sensitive Na+ channels in the kidney (29) and intestine (3), in which adenosine has been shown to be a potent regulator of their function.
Ion replacement studies demonstrated that K+ ions make a major contribution to the basal whole cell current in A549 cells. Studies from several laboratories have shown that Ca2+- and cAMP-mediated agonists regulate I K and KCNQ channels, respectively. I K channels, which are apparently absent in excitable tissues, are predominantly expressed in peripheral tissues including endothelia, epithelia, and the hematopoietic system (23, 24). These channels are thought to play a crucial role in the regulation of Cl− and HCO secretion in human airway epithelial cells (13). Activation of the basolateral cAMP-dependent K+ channel KvLQT1 (KCNQ1), in parallel with the apically located CFTR, has been shown to play an important role in maintaining cAMP-dependent Cl− secretion in human airways (30). In this study we found that XE-991, a specific inhibitor of cAMP-dependent K+ channels (40), had no effect on basal whole cell current or the subsequent response to 100 μM adenosine, suggesting that these channels do not contribute to the cell membrane conductance. In contrast, studies with an opener (1-EBIO) and a blocker (clotrimazole) ofI K channels showed that these channels are a major contributor to current. Similarly, the fact that clotrimazole abolished the current response to adenosine indicated thatI K channels were a target for adenosine action.
Epithelial nucleoside transport has been most extensively studied in intestine, kidney, liver, and choroid plexus (1, 7, 46). Enterocytes of the small intestine, for example, contain transcripts for all five of the CNT and ENT isoforms (21, 36, 37, 44,45) and express CNT1/2 functional activity in their apical membrane and ENT1 and/or ENT2 functional activity at the basolateral membrane (reviewed in Ref. 46). Cultured T84 cells, a model of intestinal crypt cells, express basolaterally restricted ENT1/2 functional activity and nucleoside uptake across the apical membrane having the characteristics of passive diffusion (32,42). Similar properties have been described for the colonic epithelial cell line Caco-2 (42), although an earlier study found these cells to express CNT3-type transport activity at the apical surface (2). Immunocytochemical analyses have demonstrated the presence of CNT1 protein in the apical membrane of rat small intestine but not at the basolateral membrane, and a similar apical localization was identified for kidney proximal tubule (20). In rat liver parenchymal cells, CNT1 was abundant in bile canalicular membranes but largely excluded from sinusoidal membranes (20), which, instead, are enriched in CNT2 immunoreactivity (15). Choroid plexus expresses CNT3-type functional activity (43). Much less is known about nucleoside transport in other epithelia, although pharmacological and RT-PCR studies suggest the presence of CNT2 but not CNT1 in rat epididymal epithelium (9).
In the present study, we have used complementary molecular and functional approaches to investigate the nucleoside transport capabilities of A549 cells. In the first series of experiments, RT-PCR was used in conjunction with isoform-specific oligonucleotide primers to test for the presence of hCNT1, hCNT2, hCNT3, hENT1, and hENT2 mRNA. In the second series, transport studies were used to confirm the identity of the expressed nucleoside transporters and to investigate their vectorial distribution (apical vs. basolateral membrane). Our results show that A549 cells lack transcripts for hCNT1, hCNT2, and hCNT3. Functionally, we also failed to detect any Na+-dependent adenosine or uridine transport activity. Thus A549 cells represent another example of an epithelial cell line lacking Na+-dependent mechanisms of nucleoside transport. Instead, RT-PCR analyses identified transcripts for hENT1 and hENT2. Both transport activities were detected in apical as well as basolateral membranes (hENT1 > hENT2). Although hENT1 and hENT2 both transport adenosine and uridine and are broadly selective for other purine and pyrimidine nucleosides, the two transporters are not functionally equivalent. For example, hENT1 has generally higher apparent substrate affinities, whereas hENT2 is also capable of interacting with nucleobases (12, 18, 19). The two transporters may therefore fulfill complementary but distinct physiological functions.
In summary, the results of this study show that both adenosine receptors and transporters control adenosine effects on K+channel function in A549 cells. Extracellularly generated adenosine is either transported via ENT1 (or, to a lesser extent, ENT2) into the cell or can activate adenosine receptors expressed on the cell surface. Inhibition of adenosine transport leads to an increase in adenosine concentration in the extracellular space and activation of adenosine receptors. Nucleoside transport may therefore represent an endogenous regulatory mechanism for adenosine-dependent control of ion secretion in human airway epithelial cells. A better understanding of this system could lead to the development of a novel therapeutic strategy in asthma and other respiratory disorders characterized by altered composition and quantity of airway surface liquid.
This work was supported by the Alberta Lung Association, the Canadian Institutes of Health Research, the Canadian Cystic Fibrosis Foundation, the National Cancer Institute of Canada (with funds from the Canadian Cancer Society), the Alberta Cancer Board, the Wellcome Trust, and the Medical Research Council of the United Kingdom.
Address for reprint requests and other correspondence: M. Duszyk, Dept. of Physiology, Univ. of Alberta, 7–46 Medical Sciences Bldg., Edmonton, AB, Canada T6G 2H7 (E-mail:).
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- Copyright © 2001 the American Physiological Society