Regulation of K+ current in human airway
epithelial cells by exogenous and autocrine adenosine
Artur J.
Szkotak1,
Amy
M. L.
Ng1,
Jolanta
Sawicka1,
Stephen A.
Baldwin2,
S. F. Paul
Man1,
Carol E.
Cass3,
James D.
Young1, and
Marek
Duszyk1
Membrane Transport Research Group, Departments of
1 Physiology and 3 Oncology, University of Alberta,
Edmonton, Alberta, Canada T6G 2H7; and 2 School of
Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2
9JT, United Kingdom
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ABSTRACT |
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+ (IK)
channels. A1 but not A2 receptor antagonists
blocked the effects of adenosine. RT-PCR studies showed that A549 cells
expressed mRNA for IK-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; nitrobenzylthioinosine; human concentrative nucleoside
transporter; human equilibrative nucleoside transporter; calcium-dependent intermediate-conductance potassium channels
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INTRODUCTION |
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, A2B
and 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.
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MATERIALS AND METHODS |
Cell culture.
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/cm2
onto Costar Snapwell inserts (0.45-µm pore size, 1-cm2
surface 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/cm2
at 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
M
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.
1C. 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 M
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 (
) was computed from the equation I =
(V
Erev), where
I 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 at V = 40 mV.

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Fig. 1.
Stimulatory effect of adenosine and nitrobenzylthioinosine (NBTI)
on whole cell current. A: representative traces showing the
activation of whole cell current by 100 µM adenosine (Ado,
n = 8). B: representative traces showing the
activation of whole cell current by 10 µM NBTI (n = 15). C: voltage protocol used for all experiments performed
in this study. D: summary of experiments done with adenosine
and NBTI showing the change in membrane chord conductance on addition
of nucleoside or nucleoside transport inhibitor. All values are
means ± SE of chord conductance density. *Statistical
significance (P < 0.01).
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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.
RT-PCR.
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 A260
units; Boehringer Mannheim) as primers. Thereafter, PCR was performed
in 20-µl reactions with the primer pairs (25 µM) described in Table
2. 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+ (IK)-1 channels] or both
(nucleoside transporters) directions by Taq
dideoxyterminator cycle sequencing with an automated DNA sequencer
(model 373A, Applied Biosystems, Foster City, CA).
Nucleoside transport.
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) 140 N-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.
Drugs.
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.
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RESULTS |
Effect of exogenous and autocrine adenosine on whole cell current
in A549 cells.
Figure 1A 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 1B 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).

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Fig. 2.
Adenosine and NBTI, acting in combination, display no additive
effects on whole cell current, suggesting a common pathway of
activation. A: representative traces showing no further
activation of whole cell current when NBTI (10 µM) is added to cells
treated with adenosine (100 µM, n = 3). B:
summary of experiments done with adenosine in combination with NBTI.
All values are means ± SE of chord conductance density.
*P < 0.05.
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Fig. 3.
CGS-15943 inhibits NBTI-stimulated whole cell current. A:
representative traces showing inhibition of 10 µM
NBTI-stimulated whole cell current with 1 µM CGS-15943
(n = 6). B: summary of experiments done with
NBTI followed by CGS-15943. All values are means ± SE of chord
conductance density. *P < 0.05.
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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).

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Fig. 4.
1-3-Dipropyl-8-cyclopentylxanthine (DPCPX), but not
3,7-dimethyl-1-propargylxanthine (DMPX), inhibits
adenosine-stimulated current. A:
representative traces showing inhibition of 100 µM
adenosine-stimulated whole cell current with 10 nM DPCPX
(n = 6). B: representative traces showing
that 10 µM DMPX does not inhibit adenosine-stimulated whole cell
current (n = 6). C: summary of experiments
performed with adenosine followed by DPCPX. D: summary of
experiments performed with adenosine followed by DMPX. All values in
C and D are means ± SE of chord conductance
density. *P < 0.05.
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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 of
IK 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 IK
channels (Fig. 5). Clotrimazole (10 µM)
inhibited basal whole cell current (P < 0.05, n = 8) and abolished the subsequent response to
adenosine, indicating that IK channels were a
likely target for adenosine action.

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Fig. 5.
Clotrimazole inhibits whole cell current and the subsequent
response to adenosine. A: representative traces showing
clotrimazole (10 µM) inhibition of whole cell current
(n = 8) and subsequent lack of response to 100 µM
adenosine (n = 3). B: summary of experiments
showing the effect of clotrimazole alone and in combination with
adenosine. Values are means ± SE of chord conductance density.
*P < 0.05.
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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 A1
receptors in A549 cells activates K+ transport, likely
through IK channels.

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Fig. 6.
Amiloride affects neither basal whole cell current nor the
subsequent response to adenosine. A: representative traces
showing no effect of amiloride (10 µM, n = 6) on
whole cell current and no subsequent alteration of response to 100 µM
adenosine (n = 4). B: summary of experiments
showing the effect of amiloride alone and in combination with adenosine
on cell membrane chord conductance density. Values are means ± SE. *P < 0.05.
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Fig. 7.
4,4'-Diisothiocyanostilbene-2,2'- disulfonic acid (DIDS)
inhibits basal whole cell current but does not prevent the subsequent
response to adenosine. A: representative traces showing DIDS
(50 µM) inhibition of whole cell current (n = 12) and
subsequent response to 100 µM adenosine (n = 12).
B: summary of experiments showing the effect of DIDS alone
compared with baseline (*P < 0.05) and DIDS in
combination with adenosine compared with DIDS alone
(**P < 0.05). Values are means ± SE of chord
conductance density.
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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.
8A, A549 cells express mRNA
for A1, A2A, and A2B but not
A3 receptors. Interestingly, studies with selective
adenosine receptor antagonists indicate that only the A1
receptor is involved in the regulation of whole cell current.

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Fig. 8.
Expression of adenosine receptors, Ca2+-dependent
intermediate-conductance K+ (IK)
channels, and nucleoside transporters, characterized by RT-PCR.
A: A549 cells express transcripts for A1,
A2A, and A2B but not A3 receptors.
B: A549 cells express IK-1 channel
mRNA. A representative positive control, using primers specific for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, is also
included. C: A549 cells express transcripts for both
human (h) equilibrative nucleoside transporter (ENT)1 and hENT2 but not
for concentrative nucleoside transporters hCNT1, hCNT2, and hCNT3.
Arrows indicate corresponding DNA marker band sizes for reference. bp,
Base pairs.
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Electrophysiological studies indicated that IK
channels function in A549 cells and are involved in the response to
adenosine. Our RT-PCR data confirm the presence of mRNA for the
IK-1 protein in A549 cells (Fig. 8B).
Figure 8C 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), IK
channels (IK-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. 10A). 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. 10B) as well as apical
and basolateral transport of uridine (Fig. 10, C and
D), 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.

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Fig. 9.
Time course of adenosine uptake across the apical and
basolateral membranes of A549 cells. Uptake of 10 µM adenosine
(pmol/mg protein) is greater across the apical membrane than the
basolateral membrane, and it is linear across both membranes for the
first 3 min. Data are means ± SE.
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Fig. 10.
Functional characterization of nucleoside uptake in A549 cells.
Initial rates of 10 µM adenosine (A, B) and
uridine (C, D) uptake (pmol · mg
protein 1 · min 1) across the apical
(A, C) and basolateral (B, D) membranes were
measured over a 2-min time course. In all experiments, nucleoside
uptake was not significantly effected by the removal of
Na+, indicating an absence of concentrative
Na+-nucleoside cotransport. Adenosine and uridine uptake
across both apical and basolateral membranes was sensitive to
inhibition by 1 µM NBTI. However, a small component of nucleoside
uptake was not inhibited by NBTI but was sensitive to inhibition by
excess unlabeled uridine (5 mM), indicating a minor contribution of
NBTI-insensitive nucleoside transport to total nucleoside
transport. The remaining nucleoside uptake was via passive
(non-transporter-mediated) mechanisms. Similar patterns were observed
for adenosine and uridine uptake, indicating broad permeant selectivity
for both NBTI-sensitive and NBTI-insensitive mediated transport in A549
cells.
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DISCUSSION |
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 A1
receptor 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 IK and KCNQ
channels, respectively. IK 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) of
IK channels showed that these channels are a
major contributor to current. Similarly, the fact that clotrimazole
abolished the current response to adenosine indicated that
IK 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.
 |
ACKNOWLEDGEMENTS |
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.
 |
FOOTNOTES |
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:
marek.duszyk{at}ualberta.ca).
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 8 May 2001; accepted in final form 17 August 2001.
 |
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