Departments of 1 Physiology, 2 Ophthalmology, and 4 Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085; and 3 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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Adenosine
stimulates Cl channels of
the nonpigmented (NPE) cells of the ciliary epithelium. We sought to
identify the specific adenosine receptors mediating this action.
Cl
channel activity in
immortalized human (HCE) NPE cells was determined by monitoring cell
volume in isotonic suspensions with the cationic ionophore gramicidin
present. The A3-selective agonist
N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide
(IB-MECA) triggered shrinkage (apparent
Kd = 55 ± 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and
A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 µM
adenosine when all four known receptor subtypes are occupied. The
A1-selective agonist
N6-cyclopentyladenosine
exerted a small effect at 100 nM but not at higher or lower
concentrations. The A2A agonist
CGS-21680 triggered shrinkage only at high concentration (3 µM), an
effect blocked by MRS-1191. IB-MECA increased intracellular
Ca2+ in HCE cells and also
stimulated short-circuit current across rabbit ciliary epithelium.
A3 message was detected in both
HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess
A3 receptors and that adenosine
can activate Cl
channels in
NPE cells by stimulating these A3 receptors.
aqueous humor secretion; chloride channels; N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide; MRS-1097; MRS-1191; MRS-1523
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INTRODUCTION |
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THE AQUEOUS HUMOR IS FORMED by the ciliary epithelium,
which comprises two cell layers: the outer pigmented cells facing the stroma and the inner nonpigmented (NPE) cells in contact with the
aqueous humor. Secretion is thought to reflect a primary transfer of
solute, principally NaCl, from the stroma into the aqueous humor, with
the secondary transfer of water down its chemical gradient. One major
factor governing the rate of secretion is the rate of
Cl release from the NPE
cells into the aqueous humor (3).
Recently, adenosine has been found to activate NPE
Cl channels that subserve
this release (2). Adenosine triggered isotonic shrinkage of cultured
human cells from the human ciliary epithelial (HCE) cell line. The
contribution of Cl
channels
to this shrinkage was identified by performing the experiments in the
presence of the cation ionophore gramicidin. In addition, adenosine
produced a Cl
-dependent
increase in short-circuit current across rabbit iris-ciliary body while
the nonmetabolizable adenosine analog 2-Cl-adenosine was shown to
activate Cl
currents in HCE
cells using the whole cell patch-clamp technique. Although this study
clearly established that adenosine could activate Cl
channels on NPE cells,
the concentrations of agonist used were capable of stimulating all four
known adenosine receptor subtypes: A1,
A2A,
A2B, and
A3 (12, 13, 25). Ciliary
epithelial cells are known to possess
A1,
A2A, and
A2B adenosine receptors (27, 35,
36). Although stimulation of these receptors can be associated with
specific changes in the levels of second messengers cAMP (6, 35, 36)
and Ca2+ (11), the effect of these
receptors on Cl
channels of
NPE cells is unknown.
The aim of the present study was to determine which receptor mediates
the activation of Cl
channels by adenosine. We now report that
A3 receptors are present on human
and rabbit NPE cells and underlie the activation of NPE Cl
channels by adenosine.
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MATERIALS AND METHODS |
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Cultured cells. We have continued to study the HCE cell line (2), an immortalized NPE cell line developed by one of us (M. Coca-Prados) from primary cultures of adult human epithelium. Cells were grown in DMEM (no. 11965-027; GIBCO BRL, Grand Island, NY) with 10% FBS (A-1115-L; HyClone Laboratories, Logan, UT) and 50 µg/ml gentamycin (no. 15750-011, GIBCO BRL), at 37°C in 5% CO2 (36). The growth medium had an osmolality of 328 mosmol. Cells were passaged every 6-7 days and were studied 8-13 days after passage, after reaching confluence.
Measurement of cell volume in isosmotic solution. A 0.5-ml aliquot of the cell suspension in DMEM was added to 20 ml of each test solution, which contained (in mM): 110.0 NaCl, 15.0 HEPES, 2.5 CaCl2, 1.2 MgCl2, 4.7 KCl, 1.2 KH2PO4, 30.0 NaHCO3, and 10.0 glucose, at a pH of 7.4 and osmolality of 298-305 mosmol. Parallel aliquots of cells were studied on the same day. One aliquot usually served as a control, and the others were exposed to different experimental conditions at the time of suspension. The same amount of solvent vehicle (dimethylformamide, DMSO, or ethanol) was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts (4).
Cell volumes of isosmotic suspensions were measured with a Coulter Counter (model ZBI-Channelyzer II), using a 100-µm aperture (4). As previously described (37), the cell volume (Vc) of the suspension was taken as the peak of the distribution function. Cell shrinkage was fit as a function of time (t) to a monoexponential function
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(1) |
Transepithelial measurements. Adult male Dutch belted rabbits weighing 1.8-2.4 kg (Ace Animals, Boyertown, PA) were anesthetized with pentobarbital sodium and killed (1). After enucleation, the iris-ciliary body was isolated as previously described (1). The experiments were in accordance with the Resolution on the Use of Animals in Research of the Association for Research in Vision and Ophthalmology.
The pupil and central iris were occluded with a Lucite disc, and the iris-ciliary body was mounted between the two halves of a Lucite chamber (1). The annulus of exposed tissue provided a projected surface area of 0.93 cm2. Preparations were continuously bubbled with 95% O2-5% CO2 for maintenance of pH 7.4 in a Ringer solution comprising (in mM): 110.0 NaCl, 10.0 HEPES (acid), 5.0 HEPES (Na+), 30.0 NaHCO3, 2.5 CaCl2, 1.2 MgCl2, 5.9 KCl, and 10.0 glucose, at an osmolality of 305 mosmol. BaCl2 (5 mM) was added to the solution to block K+ currents. The transepithelial potential was fixed at 0 mV, corrected for solution series resistance, and the short-circuit current was monitored on a chart recorder. Data were digitally acquired at 10 Hz via a DigiData 1200A converter and AxoScope 1.1 software (Axon Instruments, Foster City, CA). Automatic averaging was performed with a reduction factor of 100 to achieve a final sampling rate of six per minute.
Measurements of intracellular Ca2+. HCE cells grown on coverslips for 24-48 h were loaded with 1-5 µM fura 2-AM for 30-45 min at room temperature. The cells were subject to a postincubation interval of 20-40 min at room temperature before recording began. The coverslips were mounted on a Nikon Diaphot microscope and visualized with a ×40 oil-immersion fluorescence objective. The emitted fluorescence (510 nm) from 10-12 confluent cells was acquired at a sampling frequency of 1 Hz following excitation at 340 nm and 380 nm, and the ratio was determined with a Delta-Ram system and Felix software (Photon Technology International, Princeton, NJ).Cells were perfused with an isotonic solution consisting of (in mM) 105 NaCl, 6 HEPES (acid), 4 HEPES (Na+), 2 CaCl2, 1 MgCl2, 4 KCl, 5 glucose, and 90 mannitol, at an osmolality of 327 mosmol, pH 7.4.
The ratio of light excited at 380 nm vs. 340 nm was converted into Ca2+ concentration using the following equation (15)
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(2) |
RT-PCR assays. RNA was isolated from the HCE human NPE cell line using TRIzol reagent (GIBCO BRL). Template was synthesized in vitro from the total RNA using an RNA-PCR kit (Gene AMP; Perkin Elmer, Emeryville, Ca). The reaction mixture contained MuLV RT, an antisense primer specific for the A3 subtype of adenosine receptor, and 1-5 µg of total RNA. Primers for the human A3 receptor (accession no. X76981) were selected according to the Primer Select program (DNASTAR, Madison, WI). The forward (sense) primer (nucleotides 914-937) was 5'-GCGCCATCTATCTTGACATCTTTT-3'. The reverse (antisense) primer (nucleotides 1,373-1,355) was 5'-CTTGGCCCAGGCATACAGG-3'. The cDNA was amplified by annealing the set of oligonucleotide primers (0.2 µM) in a final volume reaction of 100 µl in an Omnigene Thermal Cycler (no. 480; HYBAID, Franklin, MA). The PCR reaction was conducted for 35 cycles, each cycle comprising 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The final extension was prolonged by 7 min at 72°C. The PCR product was reamplified using the touchdown PCR method with fresh primers and Taq polymerase, using an annealing temperature ranging from 58 to 48°C. The resulting PCR product was size-fractionated by electrophoresis on 1% agarose gel. To sequence the PCR product, a band of the expected size (462 bp) was extracted from low-melting point agarose gel using a Qiaex II Agarose Gel Extraction kit. The purified reaction product was directly sequenced on an ABI100 sequencer by the DNA Sequencing Facility at the Cell Center of the University of Pennsylvania and compared with the predicted sequence using a DNASTAR program.
The RT-PCR assay of rabbit A3 message was conducted in the same way with the following changes. RNA was obtained from the tips of New Zealand White rabbit ciliary processes using TRIzol reagent and was reverse transcribed using 3-6 µg total RNA, MuLV RT, and oligo(dT) primers. The reaction was carried out at 42°C for 30 min, followed by 5 min at 95°C. The PCR reaction and reamplification steps were performed using Amplitaq Gold (Perkin-Elmer), and 10% glycerol was included in the reamplification step. Specific primers for the rabbit A3 receptor were selected from the rabbit A3 sequence (accession no. U90718); the forward primer (nucleotides 147-167) was 5'-CAACCCCAGCCTGAAGACCAC-3', and the reverse primer (nucleotides 608-587) was 5'-TGAGAAGCAGGGGGATGAGAAT-3'. Both PCR amplification and reamplification were performed for 35 cycles, each cycle consisting of 1 min at 95°C, 1 min at 58.5°C, and 1 min at 72°C. A final extension cycle of 7 min at 72°C completed the reaction.
The product of the PCR reamplification of rabbit tissue was cloned into the PCR-TOPO vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer's directions. After transformation, plasmids were isolated using the Wizard Plus Miniprep DNA Purification System (Promega, Madison, WI). The cloned plasmid was cut with EcoR I restriction nuclease, and a band of approximately the expected size (479 bp) was identified by running the cut product on an agarose gel. The plasmid was sequenced from the Sp6 promoter site 80 bp proximal to the PCR product. The sequence was compared with the expected rabbit A3 sequence using a DNASTAR program.
Chemicals. All chemicals were reagent grade. Gramicidin and adenosine were purchased from Sigma Chemical (St. Louis, MO). N6-cyclopentyladenosine (CPA), CGS-21680, N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and 2-chloro-IB-MECA (Cl-IB-MECA) were obtained from Research Biochemicals International (Natick, MA). Fura 2-AM was bought from Molecular Probes (Eugene, OR). MRS-1097, MRS-1191, and MRS-1235 were gracious gifts from Drs. Kenneth A. Jacobson (National Institutes of Health) and Bruce L. Liang (University of Pennsylvania).
Data reduction. Values are presented as means ± SE. The null hypothesis, that the experimental and baseline measurements shared the same mean and distribution, was tested with Student's t-test and by the upper significance limits of the F-distribution, as indicated. The t-test was applied to compare the significance between single means or single fit parameters. The F-distribution was applied to test whether the time course of volume measurements in different suspensions could reflect a single population of data points.
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RESULTS |
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Human NPE cells. In previous studies
demonstrating that adenosine causes isotonic cell shrinkage by
activating Cl channels in
NPE cells (2), the levels of adenosine used were sufficiently high to
activate A1,
A2A,
A2B, or
A3 adenosine receptor subtypes
(12, 13, 25). To differentiate among these receptors, the experiments
were repeated in the present study using a series of agonists and
antagonists selective for these receptors. Because we wished to
identify the effects of these receptors specifically on
Cl
channels, 5 µM
gramicidin D was included in all solutions to eliminate any potential
contribution from K+ channels.
This ionophore readily partitions into plasma membranes to form a
cation-selective pore and is widely used for studying volume regulation
(16). Under these conditions, release of cell Cl
becomes the
rate-limiting factor in both hypo- (4) and isosmotic cell shrinkage
(2).
In the presence of gramicidin, the
A3 agonist IB-MECA caused the
cells to shrink in a concentration-dependent manner (Fig. 1, A and
B). Least-squares analysis of the
linearized Lineweaver-Burke plot generated from monoexponential fits of
these data indicates that the apparent
Kd for the
IB-MECA-induced shrinkage was 55 ± 10 nM (Fig.
1C). IB-MECA is specific for the
A3 receptor; the Ki for the
A3 receptor is 50 times lower than
it is for A1 or A2A receptor (14, 20, 21).
Cl-IB-MECA is even more specific for
A3 receptors, with a
Ki for
A3 receptors 2,500 times lower than for A1 receptors and 1,400 times lower than for A2A
receptors. The ability of Cl-IB-MECA to induce cell shrinkage (Fig.
1D) further strengthens the
hypothesis that stimulation of A3
receptors stimulates Cl
channels.
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We also tested whether we could use
A3-selective antagonists to
prevent the putative A3-mediated
shrinkage produced by IB-MECA. We preincubated parallel aliquots of
suspensions with MRS-1097, an
A3-selective antagonist with
Ki values for the
binding (in nM) to human A1,
A2, and
A3 receptors of 5,930, 4,770, and
108, respectively (22). Preincubation for 2 min with 300 nM MRS-1097 blocked the isosmotic shrinkage characteristically triggered by 100 nM
IB-MECA (Fig.
2A). We
also used a second highly selective A3 antagonist, MRS-1191 (24), with
Ki values for the
binding (in nM) to human A1,
A2, and
A3 receptors of 40,100, >100,000, and 31.4, respectively (22). Preincubation for 2 min with
100 nM MRS-1191 also prevented the subsequent response to 100 nM
IB-MECA (Fig. 2B). There was an
indication in the results of Fig. 2B
that MRS-1191 might actually produce a small amount of cell swelling. This was not a constant finding (Fig.
3B), and
may have reflected variations in the background level of
A3-receptor occupancy.
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The physiological agonist reaching the adenosine receptors is likely to be the nucleoside adenosine itself, arising from release of ATP by the ciliary epithelial cells and ecto-enzyme activity (30). We have previously found that adenosine triggers isosmotic shrinkage of cultured human NPE cells with an EC50 of 3-10 µM (2). In this concentration range, adenosine acts as a nonselective agonist of all four subtypes of the adenosine receptor (12, 13). As illustrated by Fig. 3, 2-min preincubation with either 100 nM of the A3-selective antagonist MRS-1191 (Fig. 3B) or 300 nM of the A3-selective antagonist MRS-1097 (Fig. 3A) blocked the shrinkage characteristically produced by 10 µM adenosine. MRS-1523, an A3 antagonist with Ki values for the binding (in nM) to human A1, A2, and A3 receptors of 15,600, 2,050, and 19, respectively (28), also eliminated the actions of adenosine (Fig. 3C).
The ability of specific A3
antagonists to inhibit the response to the nonspecific adenosine
suggests that the contribution of the other receptors to
Cl channel activation was
minimal. To test this further, the effects of
A1 and
A2A agonists were tested. CPA is
an A1-selective agonist with a
Ki for the
A1 receptor of 0.6 nM (31).
However, CPA produced no significant shrinkage at 30 nM and 1 µM
(data not shown, n = 3 experiments)
and 3 µM (Fig.
4A). A
small slow effect of uncertain significance was detected at the
intermediate concentration of 100 nM (Fig.
4A). Some cross-reactivity with
A3 receptors might be expected,
given the Ki of
CPA for the A3-subtype of 43 nM
(25). CGS-21680 is a widely used
A2A agonist with an
IC50 value of 22 nM for the
A2A receptor (17, 23). CGS-21680
had no detectable effect at 100-nM concentration (Fig.
4B), but did trigger isosmotic shrinkage at a 30-fold higher concentration (3 µM) (Fig.
4C). However, the
Ki for the
CGS-21680 at the A3 receptor is 67 nM (25), and thus CGS-21680 could have been acting through either A2A receptors or
A3 receptors at the higher
concentration. To distinguish between these possibilities, we
preincubated parallel aliquots of suspensions with 100 nM of the
antagonist MRS-1191. MRS-1191 prevented the shrinkage produced by the
high concentration of CGS-21680 (Fig.
4C, P < 0.01, F-test), indicating that the shrinkage observed was mediated by cross-reactivity with
A3 receptors. Because there are
presently no high-affinity A2B
agonists (25), the contribution of
A2B receptor stimulation was not
pursued, although the ability of
A3 antagonists to inhibit the
response to 10 µM adenosine (Fig. 3) argues against a role for the
A2B receptor.
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In other cells, stimulation of the
A3 receptor can lead to an
elevation of intracellular Ca2+
(26), so we monitored intracellular
Ca2+ in HCE cells to provide an
additional physiological assay for the presence of
A3 receptors. Superfusion of HCE
cells with 100 nM IB-MECA produced a sustained, repeatable, and
frequently reversible increase in the intracellular
Ca2+ concentration (Fig.
5). The increase in
Ca2+ was dependent on
concentration, with 100 nM IB-MECA leading to a mean rise of 17 ± 5 nM Ca2+
(P < 0.01, n = 8) whereas 1 µM IB-MECA
increased intracellular Ca2+ by 22 ± 6 nM (P < 0.05, n = 3). Although these changes were
relatively small, they were sustained, suggesting that these increases
in Ca2+ could be responsible for
physiological effects occurring on a time scale of minutes to hours.
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RT-PCR amplifications of RNA from the human NPE cells were conducted
using primers for the human
A3-type adenosine receptor. A
fragment of the expected 462-bp size was obtained and was enhanced by
direct PCR amplification of the product (Fig.
6). The sequence obtained from the
reamplified product was compared with the sequences of known human
adenosine receptors using the DNASTAR program. The results displayed a
97.4% similarity to the published base sequence for the
A3 receptor, whereas the
similarity indexes for the other known adenosine-receptor subtypes were
all <40% [37.9% for A1
(accession no. 68485), 35.0% for
A2A (accession no. 68486), and
36.7% for A2B (accession no.
68487)]. No product was detected when RT was excluded from the
initial reaction mixture.
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Rabbit iris-ciliary body. Adenosine in
high concentration (100 µM) has been found to increase the
short-circuit current across the rabbit ciliary body (2). We therefore
tested whether a high concentration (30 µM) of the
A3 agonist IB-MECA also affected short-circuit current. At this concentration, the vehicle
(dimethylformamide) itself exerts significant effects (Fig.
7, lowest trajectory). We corrected for the
solvent effect in the following way. Solvent alone was initially
introduced (to 0.1%), followed by the same volume of solvent (to
0.2%) containing agonist, and ending with addition of a third
identical volume of solvent alone (to a final concentration of 0.3%).
The reduction in short-circuit current following the first addition of
solvent was always greater than the third. In each of four experiments,
we averaged the time courses of the first and third additions to
estimate the effect of raising the solvent concentration without
agonist from 0.1 to 0.2% during the experimental period. Figure 7
presents the mean trajectory for the averaged solvent effect, the
uncorrected mean time course following exposure to IB-MECA, and the
mean trajectory ± SE for the solvent-corrected response. The
experiments were performed in the presence of 5 mM
Ba2+ to minimize the contribution
of K+ currents. IB-MECA produced a
significant increase in the short-circuit current; an increase in
short-circuit current in the presence of
Ba2+ suggests that the effect is
mediated by activating a Cl
conductance on the basolateral membrane of the NPE cells. The sustained
nature of the stimulation is consistent with the time course of the
cell shrinkage in response to A3
stimulation.
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In view of the short-circuit response to IB-MECA, RT-PCR amplification was also conducted with rabbit ciliary processes, using primers for the rabbit A3-type adenosine receptor. The RT-PCR product was reamplified, cloned, and sequenced. The sequence displayed a 97.4% similarity with the published base sequence for the rabbit A3 receptor. There was only 27.9% homology between rabbit A1 (accession no. L01700) and A3 receptors. Sequences are not yet available for the remaining A2A and A2B subtypes of adenosine receptors in the rabbit. Our rabbit product also displayed 75.1% similarity to the human A3 receptor but only <30% similarity indexes for the other human adenosine-receptor subtypes (28.2% for A1, 27.7% for A2A, and 29.5% for A2B). No product was detected when RT was excluded from the reaction mixture.
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DISCUSSION |
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Measurements of short-circuit current across intact rabbit ciliary
epithelium, of cell volume in suspended cultured human NPE cells, and
of whole cell currents from patch-clamped cultured human and fresh
bovine NPE cells have indicated that adenosine-receptor occupancy
stimulates Cl secretion in
mammalian NPE cells (2). The following evidence strongly suggests that
these effects are mediated by A3
receptors. A3 receptors are
present in both human HCE cells and rabbit ciliary body. The
A3-selective agonist IB-MECA
increased the short-circuit current across rabbit iris-ciliary body in
the presence of Ba2+, a change
consistent with an increased efflux of
Cl
from NPE cells. In the
presence of gramicidin to isolate the Cl
conductance, IB-MECA
caused human HCE cells to shrink in a dose-dependent manner; the
Kd of ~55 nM is
consistent with a maximal stimulation of
A3 receptors in cardiac myocytes
at 100 nM IB-MECA (34). The highly specific
A3 agonist Cl-IB-MECA also
produced shrinkage of HCE cells in the presence of gramicidin. The
A3 antagonists MRS-1097 and
MRS-1191 were able to prevent the shrinkage induced by IB-MECA at
concentrations far below their
Ki for
A1 and
A2A receptors. The
A1 agonist CPA did not have a
consistent effect on cell volume. The
A2A agonist CGS-21680 had no
effect at low concentrations. The effect of CGS-21680 on shrinkage was
only detected at a concentration 500-fold higher than the
Ki values for the
A3 receptor, and this effect was
blocked by the A3 agonist MRS-1191. The A3 antagonists
MRS-1097, MRS-1191, and MRS-1523 blocked the shrinkage produced by 10 µM adenosine; at the concentrations used, <20% of the
A1 and
A2A receptors could have been
occupied by MRS-1097 and <1% of those receptors could have been
blocked by MRS-1191 and MRS-1523. Together, these observations lead us to conclude that the adenosine-stimulated activation of
Cl
release by the HCE line
of human NPE cells is primarily mediated by occupancy of an
A3-subtype adenosine receptor.
The implications of this conclusion are subject to at least four
caveats. First, our experiments were designed to isolate the
Cl component of both the
volume and short-circuit current response. It is likely
K+ channels may also be activated
by an A3 receptor, for we
previously reported that adenosine activates a
Ba2+-sensitive component of
short-circuit current across the rabbit ciliary epithelium (2). We
would expect at least some component of this response to be mediated by
an A3 receptor, because the NPE
cells possess KCa channels (10, 18) and our present study suggests that IB-MECA elevates intracellular
Ca2+. Second, occupancy of
A1,
A2A, and
A2B receptors may well have physiologically important effects on transport mechanisms other than
Cl
channels. For example,
occupancy of A1 receptors by CPA
alters intracellular cAMP (34), and cAMP activates
K+ channels of these cells (4).
Changes in K+-channel activity can
alter membrane potential, thereby changing the electrical driving force
for secretion. Depending on the baseline level of
Cl
- and
K+-channel activity, the actions
on K+ channels could dominate the
overall response to adenosine receptor stimulation. This possibility
may be relevant to the reports that A2A receptors stimulate and
A1 receptors inhibit aqueous humor secretion in rabbits (6, 7). Third, the effect of adenosine at
concentrations other than those used here (Fig. 3) may alter the
relative contribution of the adenosine receptor subtypes to the
Cl
channel response. It
should be emphasized, however, that the concentration of adenosine used
in this study (10 µM) is likely to be physiologically relevant;
purine release from intracellular stores of ciliary epithelial cells is
expected to raise adenosine to approximately this level (30). Fourth,
the ability of IB-MECA to increase intracellular
Ca2+ provides a physiological
assay showing the existence of A3
receptors on NPE cells attached to a substrate. Further work is
required to show whether the elevation in
Ca2+ is responsible for activating
Cl
channels or whether the
A3 receptor acts synergistically
with other stimuli to further increase
Ca2+ as the
A1 receptor does (11).
The results of the present study add to a growing body of evidence suggesting that the A3 receptors, the most recently identified subtype of adenosine receptors, may have multiple important physiological functions. Pharmacological identification of these receptors has been enormously facilitated by the recent and continuing introduction of highly selective A3-receptor agonists (such as IB-MECA and Cl-IB-MECA) and antagonists (such as MRS-1097, MRS-1191, and MRS-1523). Despite these advances, a potential functional role of A2B receptors cannot be as yet excluded, in the absence of A2B-selective agonists and antagonists and without further information concerning the binding constants of A3-selective antagonists to A2B receptors. Because of this caveat, it was important to obtain molecular confirmation of the functional data, an approach that was facilitated by the availability of the sequences of the adenosine receptors for multiple species.
The need for two rounds of PCR amplification to establish identity of the A3 message in both cultured human and fresh rabbit cells suggests that the message is present in low copy number. This appears to be a general characteristic of A3 receptors. For example, Dixon et al. (8) detected A3 message only in the testis with in situ hybridization, but found widespread distribution after amplification of the message using PCR. Indeed, a similar relationship seems to exist in the ciliary epithelium, for although message was undetected by in situ hybridization (27), we have clearly shown expression of A3 message in both human and rabbit cells by amplifying with PCR. In our studies, we did not specifically address the levels of tissue protein expression. However, low binding of the A3 receptor marker 125I-labeled N6-(4-amino-3-iodobenzyl)-adenosine-5'-N-methyluronamide (125I-AB-MECA) has been found in other tissues showing robust A3 receptor-mediated responses (9, 21). Thus the possible functional importance of A3 receptors cannot be directly correlated with copy number or binding density.
The full physiological implications of
A3 receptors are just now being
clarified. A3 receptors have also
been localized to other epithelial tissues involved in
Cl transport, such as the
kidney and the lungs (8, 19). Among the effects on nonepithelial
tissue, the potential therapeutic value of
A3 preconditioning to reduce
ischemic damage is under consideration (19, 29, 34). Whether the
protective effects of A3 receptors
during cardiac ischemia are also in part mediated by changes in
Cl
transport is unknown. In
the case of the NPE cells, the present results provide additional
information in the development of a paracrine/autocrine hypothesis of
the regulation of aqueous humor secretion. Both nonpigmented and
pigmented ciliary epithelial cells have been reported to store and
release ATP, which can then be converted to adenosine through
ecto-enzyme activity (30). An increase in the
Cl
conductance is expected
to increase the rate of aqueous humor formation (3). Therefore, the
activation of Cl
channels
by adenosine acting at A3
receptors, as shown in the present study, provides a mechanism for
adenosine to elevate the production of aqueous humor. The interactions
of A3-adenosine receptors with
other adenosine and ATP receptors and their physiological significance
in aqueous humor formation remains to be tested.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Kenneth A. Jacobson (National Institutes of Health) and Bruce Liang (University of Pennsylvania) for gifts of reagents and for stimulating discussions. The compound Cl-IB-MECA (MH-C-7-08; lot no. CM-VIII-12) was provided by Research Biochemicals International as part of the Chemical Synthesis Program of the National Institute of Mental Health, contract N01MH30003.
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FOOTNOTES |
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This work was funded in part by a Respiratory Training Grant to C. H. Mitchell (HL-07027), by grants to R. A. Stone (EY-05454) and M. M. Civan (EY-10691 and EY-12213), by a Core Facilities Grant (EY-01583), all from the National Institutes of Health, and by a grant from the Paul and Evanina Mackall Foundation Trust (to R. A. Stone). R. A. Stone is a Research to Prevent Blindness Senior Scientific Investigator.
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: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, Richards Bldg., Philadelphia, PA 19104-6085.
Received 28 August 1998; accepted in final form 7 December 1998.
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REFERENCES |
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