A3 adenosine receptors regulate Clminus channels of nonpigmented ciliary epithelial cells

Claire H. Mitchell1, Kim Peterson-Yantorno1, David A. Carré1, Alice M. McGlinn2, Miguel Coca-Prados3, Richard A. Stone2, and Mortimer M. Civan1,4

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


    ABSTRACT
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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


    INTRODUCTION
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    MATERIALS AND METHODS
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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
V<SUB>c</SUB> = V<SUB>∞</SUB> + (V<SUB>0</SUB> − V<SUB>∞</SUB>) ⋅ [<IT>e</IT><SUP>−(<IT>t</IT>−<IT>t</IT><SUB>0</SUB>)/&tgr;</SUP>] (1)
where Vinfinity is the steady-state cell volume, V0 is the cell volume at the first point (t0) of the time course to be fit, and tau  is the time constant of the shrinkage. For purposes of data reduction, the data were normalized to the first time point, taken to be 100% isotonic volume. Fits were obtained by nonlinear least-squares regression analysis, permitting both Vinfinity and tau  to be variables.

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)
[Ca<SUP>2+</SUP>] = <IT>K</IT><SUB>d</SUB> ⋅ <FENCE><FR><NU>(R − R<SUB>min</SUB>)</NU><DE>(R<SUB>max</SUB> − R)</DE></FR></FENCE> <FENCE><FR><NU>S<SUB>2</SUB>f</NU><DE>S<SUB>2</SUB>b</DE></FR></FENCE> (2)
where Rmin and Rmax are the ratios of fluorescence at 340 nM vs. 380 nM in the absence of Ca2+ and in the presence of saturating Ca2+, respectively. R is the ratio measured experimentally. S2f and S2b are the fluorescences emitted at 380 nM in the Ca2+-free and Ca2+-bound states, respectively. An in situ Kd value for fura 2 of 350 nM was used (32). Rmin was obtained by bathing cells in a Ca2+-free isotonic solution containing 10 mM EGTA and 10 µM ionomycin. Rmax was obtained by bathing the cells in isotonic solution with 10 mM Ca2+ and 10 µM ionomycin. Both calibration solutions were maintained at pH 8.0 to facilitate Ca2+ exchange through ionomycin. Background fluorescence obtained from confluent HCE cells in the absence of fura 2 was subtracted from all traces. Mean values of Rmin and Rmax were used to obtain the mean responses for a set of experiments. Data were analyzed using a one-sided unpaired t-test.

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.


    RESULTS
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Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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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Fig. 1.   Concentration-response relationship for N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA)-stimulated isotonic shrinkage of nonpigmented ciliary epithelial (NPE) cells in presence of 5 µM gramicidin. In Figs. 1-4, solid trajectories are least-square fits with monoexponentials, whereas data sets displaying no significant shrinkage are connected by dotted lines. A: least-squares fits yielded the following estimated values for data obtained in parallel at concentrations of 30 nM-1 µM IB-MECA (n = 4 experiments): 30 nM [steady-state cell volume (Vinfinity ) = 98.5 ± 0.1%, tau  = 4.2 ± 1.5 min], 100 nM (Vinfinity  = 97.6 ± 0.3%, tau  = 3.5 ± 1.5 min), and 1 µM (Vinfinity  = 95.9 ± 0.3%, tau  = 2.4 ± 0.7 min). Control and 3 sets of experimental results were significantly different (P < 0.01, F-test). B: data obtained over concentration range of 1-10 µM IB-MECA (n = 4): 1 µM (Vinfinity  = 97.5 ± 0.1%, tau  = 7.2 ± 1.0 min), 3 µM (Vinfinity  = 96.7 ± 0.3%, tau  = 10.0 ± 2.3 min), and 10 µM (Vinfinity  = 97.9 ± 0.2%, tau  = 3.2 ± 1.1 min). Data obtained at 1 and 3 µM did not significantly deviate from the fit obtained with 10 µM IB-MECA. C: Lineweaver-Burk plot generated from nonlinear least-squares fits of A and B. Change in volume was calculated as (V0 - Vinfinity ). Variance with passage number was noted for tau  and (V0 - Vinfinity ). For this reason, both experimental sets (A and B) included measurements with 1 concentration (1 µM) in common. Ratio (in V0 - Vinfinity ) obtained at 1 µM in B to A was used as a scaling factor to accommodate results obtained in B with 3 and 10 µM. Using this approach, linear least-squares analysis (r2 = 0.95) led to an estimated value for the apparent Kd of 55 ± 10 nM. D: isotonic cell shrinkage was also stimulated by 100 nM Cl-IB-MECA. Fits for Cl-IB-MECA (Vinfinity  = 97.9 ± 0.2%, tau  = 2.5 ± 1.3 min) and IB-MECA (Vinfinity  = 96.8 ± 0.4%, tau  = 6.3 ± 2.2 min) are not significantly different (P > 0.05).

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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Fig. 2.   Effect of A3 antagonists on the IB-MECA-stimulated isotonic shrinkage of NPE cells. A: A3-selective antagonist MRS-1097 (300 nM) prevented shrinkage triggered by IB-MECA (P < 0.01, F-distribution). B: A3-selective antagonist MRS-1191 (100 nM) prevented characteristic shrinkage triggered by IB-MECA (n = 4, P < 0.01 by F-distribution). MRS-1191 did not affect cell volume in the absence of IB-MECA, confirming the specificity of the interaction. (n = 4).


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Fig. 3.   Effects of selective A3-receptor antagonists on adenosine-stimulated isotonic shrinkage of NPE cells. Application of 300 nM MRS-1097 (A; n = 4), 100 nM MRS-1191 (B; n = 3), and 100 nM MRS-1523 (C; n = 3) all prevented the characteristic shrinkage triggered by nonselective activation of adenosine receptors with 10 µM adenosine (P < 0.01, F-distribution).

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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Fig. 4.   Effects of adenosine-receptor agonists on isosmotic volume of NPE cells. A: A3-selective agonist IB-MECA produced prompt shrinkage at 100 nM (n = 4, Vinfinity  = 95.6 ± 0.2%, tau  = 4.5 ± 0.6 min, P < 0.01 by F-distribution). In contrast, A1-selective agonist N6-cyclopentyladenosine (CPA) had little effect at 100 nM, and none at all at 3 µM (n = 4). B: at 100 nM, A2-selective agonist CGS-21680 exerted no effect, but A3-selective agonist IB-MECA again produced shrinkage (n = 4, P < 0.01 by F-distribution). C: at high concentration (3 µM), A2-selective agonist CGS-21680 also triggered isosmotic shrinkage. However, preincubation of the cells with the selective A3 receptor antagonist MRS-1191 (100 nM) abolished this effect (n = 4, P < 0.01, F-distribution).

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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Fig. 5.   Effects of IB-MECA on level of free intracellular calcium. Concentration of intracellular Ca2+ increased steadily after application of 100 nM IB-MECA and returned to baseline levels once IB-MECA was removed. Similar increases were observed in 7 other trials. Data were obtained at a sampling rate of 1 Hz and smoothed by 21 points. Box indicates the duration of the IB-MECA application.

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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Fig. 6.   Amplified RT-PCR product. Products of PCR reamplification using A3 primers on total RNA from human NPE (HCE) cells were run on a 1% agarose gel. Lanes A and B: duplicate 40-µl aliquots of the PCR reamplification product. Band seen in lanes A and B corresponds to predicted product of 462 bp. Lane C: 40 µl of PCR reamplification product obtained when RT was omitted from initial reaction. Extreme left lane contains a 100-bp DNA ladder molecular weight marker; numbers correspond to base pairs.

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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Fig. 7.   Effect of IB-MECA on short-circuit current (Isc) across intact rabbit ciliary epithelium. As initial step in data analysis, 20-min period of baseline current just before addition of any agent was fit by linear least-squares analysis. Line generated by that analysis was extrapolated to a point 45 min beyond introduction of that agent. Each current response was subtracted from its respective extrapolated baseline to yield a common initial baseline approximating constant zero current. All recordings were placed in register relative to time of agent introduction (time 0). Records of control (solvent), IB-MECA with solvent, and IB-MECA corrected for solvent were separately averaged. IB-MECA was always added in the presence of 5 mM Ba2+ to isolate contribution of Cl- currents to the response.

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.


    DISCUSSION
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
MATERIALS AND METHODS
RESULTS
DISCUSSION
References

1.   Carré, D. A., and M. M. Civan. cGMP modulates transport across the ciliary epithelium. J. Membr. Biol. 146: 293-305, 1995[Medline].

2.   Carré, D. A., C. H. Mitchell, K. Peterson-Yantorno, M. Coca-Prados, and M. M. Civan. Adenosine stimulates Cl- channels of nonpigmented ciliary epithelial cells. Am. J. Physiol. 273 (Cell Physiol. 42): C1354-C1361, 1997[Abstract/Free Full Text].

3.   Civan, M. M. Transport by the ciliary epithelium of the eye. News Physiol. Sci. 12: 158-162, 1997.[Abstract/Free Full Text]

4.   Civan, M. M., M. Coca-Prados, and K. Peterson-Yantorno. Pathways signaling the regulatory volume decrease of cultured non-pigmented ciliary epithelial cells. Invest. Ophthalmol. Vis. Sci. 35: 2876-2886, 1994[Abstract].

5.   Civan, M. M., K. Peterson-Yantorno, M. Coca-Prados, and R. E. Yantorno. Regulatory volume decrease in cultured non-pigmented ciliary epithelial cells. Exp. Eye Res. 54: 181-191, 1992[Medline].

6.   Crosson, C. E. Adenosine receptor activation modulates intraocular pressure in rabbits. J. Pharmacol. Exp. Ther. 273: 320-326, 1995[Abstract].

7.   Crosson, C. E., and T. Gray. Characterization of ocular hypertension induced by adenosine agonists. Invest. Ophthalmol. Vis. Sci. 37: 1833-1839, 1996[Abstract].

8.   Dixon, A. K., A. K. Gubitz, D. J. S. Sirinathsinghji, P. J. Richardson, and T. C. Freeman. Tissue distribution of adenosine receptor mRNAs in the rat. Br. J. Pharmacol. 118: 1461-1468, 1996[Abstract].

9.   Dunwiddie, T. V., L. Diao, H. O. Kim, J.-L. Jiang, and K. A. Jacobson. Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus. J. Neurosci. 17: 607-614, 1997[Abstract/Free Full Text].

10.   Edelman, J. L., D. D. F. Loo, and G. Sachs. Characterization of potassium and chloride channels in the basolateral membrane of bovine nonpigmented ciliary epithelial cells. Invest. Ophthalmol. Vis. Sci. 36: 2706-2716, 1995[Abstract].

11.   Farahbakhsh, N. A., and M. C. Cilluffo. Synergistic increase in Ca2+ produced by A1 adenosine and muscarinic receptor activation via a pertussis-toxin-sensitive pathway in epithelial cells of the rabbit ciliary body. Exp. Eye Res. 64: 173-179, 1997[Medline].

12.   Fredholm, B. B., M. A. Abracchio, G. Burnstock, J. W. Daly, T. K. Harden, K. A. Jacobson, P. Leff, and M. Williams. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46: 143-156, 1994[Medline].

13.   Fredholm, B. B., M. P. Abbracchio, G. Burnstock, G. R. Dubyak, T. K. Harden, K. A. Jacobson, U. Schwabe, and M. Williams. Towards a revised nomenclature for P1 and P2 receptors. Trends Pharmacol. Sci. 18: 79-82, 1997[Medline].

14.   Gallo-Rodrigez, C., X.-D. Ji, N. Melman, B. D. Siegman, L. H. Sanders, J. Orlina, Q.-L. Pu, M. E. Olah, P. J. M. van Galen, G. L. Stiles, and K. A. Jacobson. Structure-activity relationships at A3-adenosine receptors. J. Med. Chem. 37: 636-646, 1994[Medline].

15.   Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescent properties. J. Biol. Chem. 260: 3440-3450, 1985[Abstract].

16.   Hoffmann, E., L. O. Simonsen, and I. H. Lambert. Cell volume regulation: intracellular transmission. In: Interaction of Cell Volume and Cell Function, edited by F. Lang, and D. Häussinger. Heidelberg, Germany: Springer, 1993, p. 188-248. (ACEP Series 14)

17.   Hutchison, A. J., R. L. Webb, H. H. Oei, G. R. Ghai, M. B. Zimmerman, and M. Williams. CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity. J. Pharmacol. Exp. Ther. 251: 47-55, 1989[Abstract].

18.   Jacob, T. J. C., and M. M. Civan. Role of ion channels in aqueous humor formation. Am. J. Physiol. 271 (Cell Physiol. 40): C703-C720, 1996[Abstract/Free Full Text].

19.   Jacobson, K. A. Adenosine A3 receptors: novel ligands and paradoxical effects. Trends Pharmacol. Sci. 19: 184-190, 1998[Medline].

20.   Jacobson, K. A., H. O. Kim, S. M. Siddiqi, M. E. Olah, G. L. Stiles, and D. K. J. E. von Lubitz. A3-adenosine receptors: design of selective ligands and therapeutic prospects. Drugs Future 20: 689-699, 1995.

21.   Jacobson, K. A., O. Nikodijevic, D. Shi, C. Gallo-Rodriguez, M. E. Olah, G. L. Stiles, and J. W. Daly. A role for central A3-adenosine receptors. Mediation of behavioral depressant effects. FEBS Lett. 336: 57-60, 1993[Medline].

22.   Jacobson, K. A., K.-S. Park, J.-L. Jiang, Y.-C. Kim, M. E. Olah, G. L. Stiles, and X.-D. Ji. Pharmacologic characterization of novel A3 adenosine receptor-selective antagonists. Neuropharmacology 36: 1157-1165, 1997[Medline].

23.   Jarvis, M. F., R. Schulz, A. J. Hutchison, U. H. Do, M. A. Sills, and M. Williams. [3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain. J. Pharmacol. Exp. Ther. 251: 888-893, 1989[Abstract].

24.   Jiang, J.-l., A. M. van Rhee, N. Melman, X.-D. Ji, and K. A. Jacobson. 6-Phenyl-1,4-dihydropyridine derivatives as potent and selective A3 adenosine receptor antagonists. J. Med. Chem. 39: 4667-4675, 1996[Medline].

25.   Klotz, K.-N., J. Hessling, J. Hegler, C. Owman, B. Kull, B. B. Fredholm, and M. J. Lohse. Comparative pharmacology of human adenosine receptor subtypes: characterization of stably transfected receptors in CHO cells. Naunyn Schmiedebergs Arch. Pharmacol. 357: 1-9, 1998[Medline].

26.   Kohno, Y., X.-D. Ji, S. D. Mawhorter, M. Koshiba, and K. A. Jacobson. Activation of A3 adenosine receptors on human eosinophils elevate intracellular calcium. Blood 88: 3569-3574, 1996[Abstract/Free Full Text].

27.   Kvanta, A., S. Seregard, S. Sejersen, B. Kull, and B. B. Fredholm. Localization of adenosine receptor messenger RNAs in the rat eye. Exp. Eye Res. 65: 595-602, 1997[Medline].

28.   Li, A.-H., S. Moro, N. Melman, X.-D. Ji, and K. A. Jacobson. Structure-function relationships and molecular modeling of 3,5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists. J. Med. Chem. 41: 3186-3201, 1998[Medline].

29.   Liang, B. T., and K. A. Jacobson. A physiological role of the adenosine A3 receptor: sustained cardioprotection. Proc. Natl. Acad. Sci. USA 95: 6995-6999, 1998[Abstract/Free Full Text].

30.   Mitchell, C. H., D. A. Carré, A. M. McGlinn, R. A. Stone, and M. M. Civan. A release mechanism for stored ATP in ocular ciliary epithelial cells. Proc. Natl. Acad. Sci. USA 95: 7174-7178, 1998[Abstract/Free Full Text].

31.   Moos, W. H., D. S. Szotek, and R. F. Bruns. N6-cycloalkyladenosines. Potent, A1-selective adenosine agonists. J. Med. Chem. 28: 1383-1384, 1985[Medline].

32.   Negulescu, P. A., and T. E. Machen. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 192: 38-81, 1990[Medline].

33.   Shahidullah, M., and W. S. Wilson. Mobilization of intracellular calcium by P2Y2 receptors in cultured, non-transformed bovine ciliary epithelial cells. Curr. Eye Res. 16: 1006-1016, 1997[Medline].

34.   Stambaugh, K., K. A. Jacobson, J. L. Jiang, and B. T. Liang. A novel cardioprotective function of adenosine A1 and A3 receptors during prolonged simulated ischemia. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H501-H505, 1997[Abstract/Free Full Text].

35.   Wax, M. B., and R. V. Patil. Immunoprecipitation of A1 adenosine receptor-GTP-binding protein complexes in ciliary epithelial cells. Invest. Ophthalmol. Vis. Sci. 35: 3057-3063, 1994[Abstract].

36.   Wax, M. B., D. M. Sanghavi, C. H. Lee, and M. Kapadia. Purinergic receptors in ocular ciliary epithelial cells. Exp. Eye Res. 57: 89-95, 1993[Medline].

37.   Yantorno, R. E., M. Coca-Prados, T. Krupin, and M. M. Civan. Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body. Exp. Eye Res. 49: 423-437, 1989[Medline].


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