Similarity of A3-adenosine and swelling-activated Clminus channels in nonpigmented ciliary epithelial cells

David A. Carré1, Claire H. Mitchell1, Kim Peterson-Yantorno1, Miguel Coca-Prados2, and Mortimer M. Civan1,3

Departments of 1 Physiology and 3 Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104; and 2 Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, Connecticut 06510


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl- transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A3-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-beta -D-ribofuranuronamide (IB-MECA) stimulated currents (IIB-MECA) by ~90% at +80 mV. Partial replacement of external Cl- with aspartate reduced outward currents and shifted the reversal potential (Vrev) from -23 ± 2 mV to -0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl- replacement with aspartate and NO3-, and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents (ISwell). Partial cyclamate substitution for external Cl- inhibited inward and outward currents of both IIB-MECA and ISwell. Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A3-subtype adenosine agonists and swelling activate a common population of Cl- channels.

aqueous humor secretion; anion selectivity; cyclamate; 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-beta -D-ribofuranuronamide; MRS-1523


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE AQUEOUS HUMOR OF THE EYE is secreted by the bilayered ciliary epithelium (9). The consensus view (4, 5, 13, 19, 20, 31, 34, 35) is that ions are taken up from the stroma of the ciliary processes by the pigmented ciliary epithelial cells, passed through gap junctions (8, 13, 15, 23, 25, 28) to the nonpigmented ciliary epithelial (NPE) cell layer, and then released into the aqueous humor. The water is thought to passively follow the movement of these ions, mainly Na+ and Cl-, across the epithelial tissue. One major factor likely to limit the rate of secretion is the rate of release of Cl- into the aqueous humor (6). Although the regulatory pathways of Cl- channel activity are unclear, adenosine has been found to activate NPE Cl- channels (3). The potential importance of purinergic regulation is supported by the association observed between activation of A2-adenosine and A1 receptors with ocular hypertension (11) and hypotension (10), respectively.

The physiological source of the external adenosine is likely to be the ciliary epithelial cells themselves. NPE cells display reservoirs of ATP both in culture and in the intact tissue, release ATP to the extracellular fluid, and can metabolize the ATP to adenosine by their ectoenzymes (21). Adenosine and its metabolite inosine can rise to micromolar levels in the aqueous humor (12), a concentration adequate for activating Cl- channels (3).

The signaling pathway involved in adenosine activation of Cl- channels is unknown. Recently, volumetric measurements of cultured human NPE (HCE) cells in isotonic suspensions, coupled with measurements of short-circuit current across rabbit ciliary epithelium and RT-PCR amplification of RNA from cells and tissue, have suggested that adenosine activates Cl- efflux in NPE cells by stimulating A3-subtype adenosine receptors (22). We have now studied the same human nonpigmented ciliary epithelial cell line with whole cell patch clamping to examine whether Cl- channels are indeed activated by the A3 agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-beta -D-ribofuranuronamide (IB-MECA). We have also compared the anionic selectivity of IB-MECA-activated and swelling-activated Cl- channels. The results are consistent with the concept that A3 agonists and swelling activate a common population of Cl- channels.


    MATERIALS AND METHODS
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INTRODUCTION
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RESULTS
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Cellular model. The HCE-immortalized nonpigmented ciliary epithelial cell line was developed by M. Coca-Prados from primary cultures of human epithelium. Cells were grown in DMEM (no. 11965-027; GIBCO BRL, Grand Island, NY) containing 10% fetal bovine serum (A-1115-L; HyClone Laboratories, Logan, UT) and 50 µg/ml gentamycin (no. 15750-011, GIBCO BRL) at 37°C in 5% PCO2 (37). The medium had an osmolality of 328 mosmol/kgH2O. Cells were passaged every 6-7 days and were studied within 6-10 days of passage once confluence was reached.

In preparation of patch clamping, culture flasks were briefly trypsinized (37) for 4.0-5.5 min. Cells were resuspended in DMEM, plated onto the base of a plastic petri dish, and allowed to settle and adhere for 15-20 min. A perforated lucite insert was placed inside the petri dish to form the chamber contours and restrict the fluid volume to ensure rapid bath exchange.

Solutions. The compositions of the internal filling solution of the micropipette and the external bath solutions are presented in Tables 1 and 2. The internal solution contained 0.43 mM CaCl2 and 5.0 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to fix the free Ca2+ concentration at 10 nM. External and internal solutions were adjusted to pH 7.4 and 7.2, respectively, by addition of either NaOH or N-methyl-D-glucamine (NMDG). The osmolalities of the isotonic bath and internal solution were set at 295 ± 3 mosmol/kgH2O and 272 ± 3 mosmol/kgH2O, respectively. The hypotonic bath solutions were made 60 mosmol/kgH2O lower than the isotonic external solutions by suitable exclusion of mannitol.

                              
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Table 1.   Solutions for IB-MECA experiments


                              
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Table 2.   Solutions for hypotonic experiments

Ruptured-patch whole cell recording. Micropipettes were double-pulled from Corning glass (no. 7052), coated with Sylgard, and fire polished. The resistances of the micropipettes in the bath ranged from 1.0 to 2.2 MOmega ; successful seals commonly displayed resistances of 20-50 GOmega . After rupture of the membrane patch, the series resistance was measured to be 7.3 ± 0.8 MOmega . All series resistances in excess of 5 MOmega were 80% compensated. In the baseline NaCl-Ringer solutions of Tables 1 and 2, the baseline whole cell currents were 51 ± 14 pA/pF and 46 ± 13 pA/pF, respectively.

All patches were formed in the high-Cl--containing bathing solutions (NaCl-Ringer solutions) of Tables 1 and 2. The ambient potentials in the bath were nulled to 0 mV before approaching the cell. The junction potentials for the micropipettes relative to a flowing 3 M KCl junction were measured to be 4.12 ± 0.02 mV and 2.76 ± 0.02 mV for the NaCl-Ringer solutions of Tables 1 and 2, respectively. Additionally, account has been taken of the small change in junction potential arising at the 3 M KCl reference junction when the bath solution was changed after formation of the patches. Single junction potentials cannot be measured but can be estimated with the Henderson equation in the outside bath and in impaled cells (24). These calculations were conducted with the Clampex 8.0 (Axon Instruments) software program based on the approach of Barry and Lynch (2). The relative mobility of cyclamate to K+ was estimated to be 0.24 from a comparison of the measured junction potentials in the presence of NaAsp- and NaCycl-Ringer solutions, using the Clampex 8.0 program. The final corrections for junction potentials ranged from 1.5 to 4.8 mV. These corrections have been applied in the analysis of the measurements (as indicated in RESULTS and DISCUSSION) but are not included in the figures, which present the raw data.

Data were acquired at 1.25 kHz and filtered at 500 Hz with an Axopatch 1D patch-clamp amplifier (Axon Instruments, Foster City, CA). The membrane potential was held at 0 mV and usually stepped to test voltages from -100 to +80 mV in 20-mV increments at 1-s intervals (see Figs. 7 and 11). Each step lasted 819.2 ms with intervening periods of 180.8 ms at the holding potential. A briefer voltage protocol was also used (3), stepping the membrane potential to a smaller number of values (-20, ± 40, and ± 80 mV).

Results are presented in three formats, namely, the short-term time dependence of whole cell currents during the course of the voltage steps, the long-term time course of the currents before, during, and after experimental perturbations, and the current-voltage relationships (I-V). The short-term time traces (see Figs. 7 and 11) are raw currents obtained during a stable recording interval associated with a given solution. The data points of the long-term time courses (see Figs. 1-4, 8, 9A, and 10A) are the means of five consecutive samples within a given voltage step, obtained immediately after the decay of any uncompensated capacitative transient (from 17.6 to 25.6 ms after onset of the voltage step). The I-V of Fig. 9B and Fig. 10B were generated by averaging the results of 3-15 experiments. The error bars present 1 SE of the mean.

The quantification of results was complicated by the frequent steady increase in currents throughout the experiments, and several methods were used to obtain values. The stimulation attributed to IB-MECA was determined by subtracting the current values just before addition of IB-MECA from values at a plateau, or the current before application of blockers, because these drugs were not always reversible. For example, in Fig. 1, the plateau before application of 100 µM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was taken as the maximum current in IB-MECA. This method is clearly dependent on the time following application of IB-MECA, and this variability is reflected in the SE (see RESULTS). The slow, steady increase made the concentration-response experiments particularly difficult to analyze (Fig. 2). For these experiments, the rate of increase obtained using one concentration was extrapolated, and the response due to the next concentration was defined as the difference between the extrapolated value, due to the low concentration, and the observed value was attributed to the new, higher concentration. The selectivity measurements were more precise because they involved currents obtained at approximately the same time; to control for rising current, the mean value before and after Cl- substitution was obtained, and the difference currents were produced by subtracting the currents 4-5 cycles after application of the Cl--substituted solution from this mean value (see Fig. 9A). In the case of the blockers NPPB and niflumic acid, only the current immediately preceding drug application was used to control for the potential partial irreversibility of these drugs.


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Fig. 1.   Time course of effects of 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-beta -D-ribofuranuronamide (IB-MECA) on whole cell currents. Each data point represents a mean of 15 current measurements at 4-ms intervals, beginning 840 ms after the onset of the voltage step. Positive outward current is presented upwards. The command voltages were ±80 and ±40 mV, with a holding potential of 0 mV. The cell was perfused with a NaCl-Ringer solution (Table 1) except for the transient perfusion with a low Cl--containing (L) solution (NaAsp, Table 1). Currents were activated by 100 nM IB-MECA after a lag time of ~2 min. Outward currents were reduced by lowering the external Cl- concentration, and outward and inward currents were reversibly inhibited by either 10 µM (n) or 100 µM (N) 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). For purposes of clarity in this initial figure, only a subset of the 10 voltage steps has been included.



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Fig. 2.   Concentration dependence of IB-MECA-stimulated currents. Current was unaffected by 10 nM IB-MECA, and 30 nM produced only a small effect, whereas a clear increase followed application of 100 nM IB-MECA. When added after current activation, 100 nM MRS-1523 + 100 nM IB-MECA did not reduce current levels, although currents increased at a faster rate once the MRS-1523 was removed. Current was clearly inhibited by both 500 µM niflumic acid and 100 µM NPPB.

Volumetric measurements and analysis. After a single T-75 flask was harvested by trypsinization (36), a 0.5-ml aliquot of the cell suspension in DMEM (or in Cl--free medium, where appropriate) was added to 20 ml of each test solution. The standard test solution contained (in mM) 110 NaCl, 15 HEPES, 2.5 CaCl2, 1.2 MgCl2, 4.7 KCl, 1.2 KH2PO4, 30 NaHCO3, and 10 glucose, at a pH of 7.4 and osmolality of 298-305 mosmol/kgH2O. The Cl--free solution comprised (in mM) 110 sodium methanesulfonate, 15 HEPES, 2.5 calcium methanesulfonate, 1.2 MgSO4, 4.7 potassium methanesulfonate, 1.2 KH2PO4, 30 NaHCO3, and 10 glucose, at a pH of 7.4 and osmolality of 294-304 mosmol/kgH2O. 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) was always added to the control and experimental aliquots. The sequence of studying the suspensions was varied to preclude systematic time-dependent artifacts (7).

Cell volumes of isosmotic suspensions were measured with a Coulter Counter (model ZBI-Channelyzer II), using a 100-µm aperture. As previously described [Yantorno et al. (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 the simple exponential function
V<SUB>c</SUB><IT>=</IT>(V<SUB><IT>0</IT></SUB><IT>−</IT>V<SUB><IT>∞</IT></SUB>)<IT>·</IT>(<IT>e<SUP>−t/&tgr;</SUP></IT>)<IT>+</IT>V<SUB><IT>∞</IT></SUB> (1)
where Vinfinity is the steady-state cell volume, V0 is cell volume at t = 0, 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. The baseline isotonic value is ~2,000 fl. Fits were obtained by nonlinear least-squares regression analysis, permitting both Vinfinity and tau  to be variables (3).

Chemicals. All chemicals were reagent grade. IB-MECA and Cl-IB-MECA were obtained from Research Biochemicals International (Natick, MA), NPPB was purchased from Biomol Research Laboratories (Plymouth Meeting, PA), niflumic acid was purchased from Sigma (St. Louis, MO), and MRS-1523 was a gift from Dr. Kenneth Jacobson (National Institutes of Health).

Statistics. Values are presented as the means ± 1 SE. The symbol n indicates the number of experiments, except in Fig. 1, where it represents the addition of 100 µM NPPB. The probability (P) of the null hypothesis was tested with Student's two-tailed t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Stimulation of A3 receptor. The selective A3 receptor agonist IB-MECA (100nM) (14, 17) activated whole cell currents in human NPE cells (Fig. 1). An increase in current was initially detected 1-4 min after application of IB-MECA, and we have estimated that IB-MECA induced an 88 ± 31% rise in currents over baseline (n = 13). Because the current rose continually in some experiments, and in these cases the measurement of maximum current is time dependent, this is only a rough measurement (see METHODS). However, the value (and its variability) is comparable to the increases produced by a 100-fold higher concentration of adenosine (199 ± 54%; n = 4) and the nonmetabolizable analog 2-chloroadenosine (136 ± 45%; n = 10) (3).

Previous studies have shown that adenosine reduced cell volume in these NPE cells primarily by activating the A3-receptor subtype (22). Because IB-MECA is 50-fold more selective for A3 receptors than for A1 or A2a receptors (14, 16, 17), the ability of IB-MECA to stimulate currents in the present study suggests that A3 receptors are also involved and that the volume response and current stimulation may share a common mechanism. Several experiments were performed to determine the specific role of the A3 receptor and whether the volume response and current stimulation are linked. First, the concentration dependence of the current activation was examined; 10 nM IB-MECA did not produce a significant increase in current (n = 3), 30 nM produced a small, 19 ± 9% rise in current (n = 3), and in two experiments, 1 µM IB-MECA did not produce any larger response than 100 nM (Fig. 2). This is similar to the concentration-dependent action of IB-MECA on cell volume, where the dissociation constant (Kd) was 55 nM (22). Next, the specificity of the current was examined using the highly selective A3 agonist Cl-IB-MECA, which has an inhibitory constant (Ki) for A3 receptors >1,400 times lower than for A1 or A2a receptors (16). Cl-IB-MECA clearly activated currents in duplicate experiments (Fig. 3), and the ability of 10 nM to induce a response is consistent with the fact that Cl-IB-MECA has a lower Ki at the A3 receptor than IB-MECA. The specificity of the response for the A3 receptor was further shown with MRS-1523, which antagonizes A1, A2, and A3 receptors with a Ki (in nM) of 15,600, 2,050, and 19, respectively (18). In two experiments, MRS-1523 prevented activation of whole cell currents by IB-MECA when the two substances were presented simultaneously. The minimal inhibition observed when MRS-1523 was presented after the response to IB-MECA was underway (Fig. 2) is consistent with its block at the receptor level. Occasionally, the response desensitized, and increasing the concentration of A3 agonist (in this case adenosine) did not produce additional increase in current (Fig. 4A). The response was clearly uncontaminated by P2 ATP receptor contribution, because addition of ATP induced an increased response of inward currents even when the A3 receptor was desensitized (Fig. 4B).


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Fig. 3.   Activation of current by Cl-IB-MECA. The rate of increase was increased by 30 nM and 100 nM Cl-IB-MECA, but addition of 1 µM Cl-IB-MECA produced no further increase. Current was blocked by both 500 µM niflumic acid (Nif) and 100 µM NPPB.



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Fig. 4.   Response does not involve ATP receptor. A: the response occasionally desensitized, and application of the A3 agonist adenosine (Aden), even at higher concentrations, did not elicit a response. Iso, isotonic central solution. B: even after the A3 receptors had desensitized to 1 mM adenosine, addition of 1 mM ATP activated currents at hyperpolarized potentials, indicating that different receptors were involved. This trace is a continuation of the experiment in A. The decrease in current at depolarized potentials probably represents the block of outward pICln Cl- currents by ATP.

Role of Cl- channels. The reduction in cell volume by A3-receptor stimulation was found to involve the movement of Cl- from the NPE cells (22), so we asked whether the current activated by IB-MECA was carried by Cl- ions. Several observations suggest that the response does involve Cl- channels. Lowering the external Cl- concentration from 140 (NaR solution) to 25 mM (NaAsp solution) by equimolar aspartate substitution (Table 1) dramatically reduced the outward currents during IB-MECA perfusion but had little effect on the inward whole cell currents (Fig. 1). This is consistent with the movement of Cl- ions because the outward currents largely reflect entry of Cl- from the bath, whereas inward currents largely reflect outward movement of the intracellular Cl- at a fixed concentration throughout the experiment.

The pharmacological profile also implicates the activation of Cl- channels. At 100 µM, the Cl- channel blocker NPPB nearly abolished both outward and inward currents activated by IB-MECA (Figs. 1 and 2) and by Cl-IB-MECA (Fig. 3). NPPB is known to block K+ as well as Cl- channels (26), but K+ was omitted from the internal and external solutions for just this reason (Tables 1 and 2). At a concentration of 10 µM, NPPB is thought to be free of nonspecific effects (32). As displayed in Fig. 1, NPPB exerted qualitatively similar effects at 10 and 100 µM. Niflumic acid, which also blocks Cl- channels (33), showed a similar inhibition when applied to IB-MECA-activated (Fig. 2) and Cl-IB-MECA-activated currents (Fig. 3) at a concentration of 500 µM. To further strengthen the link between the reduction in cell volume and the activation of Cl- channels by A3-receptor stimulation, the effect of NPPB on cell shrinkage was tested. IB-MECA (100 nM) led to a reduction in cell volume that was inhibited by 100 µM NPPB (Fig. 5).


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Fig. 5.   Block of cell shrinkage by NPPB. IB-MECA (100 nM) led to a reduction in the volume of nonpigmented ciliary epithelial cells (Vinfinity  = 95.1 ± 0.3, tau  = 2.9 ± 0.7 min) that was blocked by 100 µM NPPB. The block appeared to be delayed, taking effect only 4 min after simultaneous presentation of both IB-MECA and NPPB.

As illustrated by Fig. 6, the reversal potential (Erev) for the IB-MECA-activated currents was well estimated by nonlinear least-squares fit (SigmaPlot) to a form of the Goldman equation and was always used to calculate the reversal potentials
&Dgr;<SUB>IB-MECA</SUB><IT>=</IT>(<IT>P</IT><SUB>Cl</SUB><IT>·F</IT>)<IT>·&bgr;·</IT>[Cl<SUP><IT>−</IT></SUP>]<SUB>o</SUB><IT>·</IT>(<IT>e<SUP>&bgr;</SUP>−e</IT><SUP><IT>&bgr;</IT><SUB>rev</SUB></SUP>)<IT>/</IT>(<IT>e<SUP>&bgr;</SUP>−1</IT>) (2)
where
&bgr;≡(V<SUB>m</SUB><IT>·F</IT>)<IT>/</IT>(<IT>R·T</IT>) (3)

&bgr;<SUB>rev</SUB><IT>≡</IT>(<IT>E</IT><SUB>rev</SUB><IT>·F</IT>)<IT>/</IT>(<IT>R·T</IT>) (4)
and Vm is the membrane potential, PCl is the Cl- permeability, F is the Faraday's constant, [Cl-]o is the external Cl- concentration, R is the perfect gas constant, and T is the absolute temperature. Estimates for both unknown parameters (PCl and Erev) were generated by the analysis. With the use of this approach, the reversal potential (corrected for junction potential) for the IB-MECA-activated currents in the NaCl-Ringer solution was -27.0 ± 1.9 mV (n = 13). No ion other than Cl- could account for this value of reversal potential (Table 1). The NPPB difference currents, obtained by subtracting values during the NPPB block from the IB-MECA-stimulated values immediately proceeding drug application, displayed a corrected reversal potential of -30.2 ± 2.7 mV. These estimates of reversal potential are less than the theoretical Nernst potential for Cl- (-43.5 mV) and could reflect the contribution of aspartate movement out of the micropipette. As discussed below, the differences from ideal Nernst behavior could be accommodated by a relative permeability for aspartate to Cl- (PAsp/PCl) of 0.18-0.23.


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Fig. 6.   Goldman fit of the IB-MECA-activated difference currents observed in NaCl-Ringer solution (Table 1). The figure presents a representative example (continuous trajectory) of the fits generated with the Goldman equation. Nonlinear least-squares fit to Eq. 1 generated values for PCl and Vm of 13.5 ± 0.5 fl/s and -22.9 ± 1.9 mV, respectively. From the mean cell capacitance of this series (32.4 ± 6.2 pF) and taking the capacitance/area ratio to be 1 µF/cm2, the value generated for PCl can also be expressed as 4.2 ± 0.8 × 10-7 cm/s.

Figure 7 presents the time courses of difference currents after step changes in voltage. The IB-MECA-stimulated difference currents showed similar outward rectification in both high- (Fig. 7A) and low-Cl- (Fig. 7, C and D) containing solutions, and inactivation at positive potentials. A similar pattern was displayed for the currents blocked by NPPB in high-Cl- solution (Fig. 7B). Comparable outward rectification and inactivation was seen in the NPPB-inhibited currents (Fig. 7B) and in currents activated by nonspecific adenosine-receptor agonists (Fig. 7; Ref. 3).


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Fig. 7.   IB-MECA and NPPB difference currents as functions of time following voltage steps. The IB-MECA difference currents were generated by subtracting the currents in the control period from the corresponding currents during the response to the A3 agonist in NaCl (A), NaAsp (C), and NaCycl (D) (see Table 1). The NPPB difference currents were measured as the NPPB-sensitive currents (B) in the presence of 100 nM IB-MECA in NaCl-Ringer solution. Here and in Fig. 11, the traces represent currents obtained from -100 mV through +80 mV in 20-mV steps, with the top trace in each figure showing the +80 mV trace.

Effects of ionic composition on response to IB-MECA. One of the identifying characteristics of Cl- channels is their anionic selectivity. For example, Voets et al. (30) differentiated between one channel associated with injection of message for the human volume-sensitive Cl- conductance regulatory protein pICln and another endogenous volume-activated channel, in part by a striking difference in the relative permeabilities to Cl-, NO3-, and cyclamate. Human pICln was first cloned in the cell line currently studied (1), and these cells also display swelling-activated Cl- channels (36). We have, therefore, examined the relative permeabilities to Cl-, NO3-, aspartate, and cyclamate, both of IB-MECA-activated and swelling-activated Cl- channels in these cultured human NPE cells.

Before the relative anionic permeability was calculated, the cation selectivity of the IB-MECA-stimulated current was determined by alternately perfusing the cell with the NaCl, NaAsp, NMDG-Cl, and NMDG-Asp Ringer solutions of Table 1. Figure 8 demonstrates that the effect of substituting NMDG for Na+ was very small before and after IB-MECA was added. The striking observation was the dependence of the whole cell currents on external Cl-. The corrected reversal potential for IB-MECA-activated channels in the presence of NMDG-Cl solution (Table 1) was -28.4 mV, almost identical with that in NaCl-Ringer solution (-27.0 mV, Table 3). Thus the IB-MECA-activated channels cannot distinguish between Na+ and NMDG in contrast to their discrimination between Cl- and aspartate.


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Fig. 8.   Time course of effects of IB-MECA as a function of dominant external cation. Each data point represents a mean of 5 current measurements at 1.6-ms intervals, beginning 17.6 ms after the onset of the voltage step. The holding potential was 0 mV, and command voltages were stepped from +80 to -100 mV in 20-mV decrements. During the control period, substitution of N-methyl-D-glucamine (NMDG)+ for Na+ had little effect on the whole cell currents, but reduction of the external Cl- by aspartate (Asp) replacement markedly lowered the outward currents. The same observations were noted after stimulation with IB-MECA, and all currents were markedly inhibited by 100 µM NPPB added at the conclusion of the experiment.


                              
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Table 3.   Estimates of reversal potentials for IB-MECA-activated currents

The anion selectivity was explored by perfusing the same cell with the Na+ salt of different anions (Fig. 9A). Both before and during perfusion with IB-MECA, partial Cl- replacement with aspartate reduced the outward positive currents. Cyclamate reduced the outward currents even more, and in addition lowered the inward currents (more easily appreciated in Fig. 9C). The application of NPPB nearly abolished inward and outward currents. Figure 9, B and C, presents the averaged reduced data in the form of I-V relationships. The IB-MECA-activated difference currents were similar in NaCl- and NaNO3-Ringer solutions, and both were also similar to the NPPB difference currents measured in the presence of IB-MECA in NaCl-Ringer solution (Fig. 9B). The corrected reversal potentials (Table 3) for these difference currents were -27 mV (NaCl), -27.6 mV (NaNO3), and -30.2 mV (NPPB in NaCl).


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Fig. 9.   Effects of IB-MECA as a function of the dominant external anions. A: perfusion with 100 nM IB-MECA increased the total currents and Cl--dependent fractions of those currents. Partial replacement of Cl- with cyclamate (Cycl) (but not aspartate) produced a small but clear inhibition of inward current only after activation by IB-MECA. NPPB produced a nearly complete, reversible inhibition of inward and outward currents. The Na+ form of the salts was used. The voltage protocol and analysis procedure of Fig. 7 were applied. B: difference currents for cells perfused with NaCl- and NaNO3-Ringer solutions, and NPPB difference currents for stimulated cells following IB-MECA activation. C: difference currents for cells perfused with NaAsp- and NaCycl-Ringer solutions for cells activated by IB-MECA.

Approximate values of the relative permeabilities can be calculated for aspartate (PAsp/PCl) and nitrate (PNO3/PCl) from the reversal potentials of Table 3, using the Goldman equation
(P<SUB>Asp</SUB><IT>/P</IT><SUB>Cl</SUB>)<IT>=</IT>{[(<IT>e</IT><SUP><IT>&bgr;</IT><SUB>rev</SUB></SUP>)<SUB>NaCl</SUB><IT>·140</IT>]<IT>−</IT>[<IT>25</IT>]}<IT>/</IT>(<IT>110</IT>) (5)

(P<SUB>NO3</SUB><IT>/P</IT><SUB>Cl</SUB>)<IT>=</IT>{[(<IT>25</IT>)<IT>+</IT>(<IT>P</IT><SUB>Asp</SUB><IT>/P</IT><SUB>Cl</SUB>)<IT>·</IT>(<IT>110</IT>)] (6)

<IT>·</IT>[(<IT>e</IT><SUP><IT>−&bgr;</IT><SUB>rev</SUB></SUP>)<SUB>NaNO3</SUB>]<IT>−</IT>(<IT>25</IT>)]}<IT>/</IT>(<IT>115</IT>)
The calculated values for (PAsp/PCl) and (PNO3/PCl) through the IB-MECA-activated channels are 0.23 and 1.03, respectively.

NaAsp-Ringer solution reduced the outward currents and shifted the corrected reversal potential to -3.1 mV. The inward currents were also slightly reduced during the course of NaAsp perfusion (compare Fig. 9, B and C). This reduction in inward currents was much more marked during partial cyclamate substitution for Cl- (Fig. 9C). The cyclamate replacement for Cl- also markedly reduced the outward currents. The possible differential effects of cyclamate and aspartate on the inward currents were examined by attempting to scale the difference currents in NaAsp (Delta INaAsp) and NaCycl (Delta INaCycl) to those in NaCl (Delta INaCl) with the scaling factors kAsp and kCycl, respectively
&Dgr;I<SUB>NaCl</SUB><IT>=k</IT><SUB>Cycl</SUB><IT>·&Dgr;I</IT><SUB>NaCycl</SUB> (7)

&Dgr;I<SUB>NaCl</SUB><IT>=k</IT><SUB>Asp</SUB><IT>·&Dgr;I</IT><SUB>NaAsp</SUB> (8)
Over the depolarizing voltage range (Vm > 0 mV), Delta INaCl was well fit by Eq. 7 with kCycl = 11.8 ± 0.3, and with kCycl = 10.7 ± 0.9 over the hyperpolarizing domain. The difference between these values of the scaling factor (1.1 ± 0.9) was not statistically significant. In contrast, the values generated for kAsp with Eq. 8 over the hyper- and depolarizing ranges differed by 0.7 ± 0.2 (P < 0.02). These calculations indicated that external cyclamate produced a voltage-independent reduction in the IB-MECA-activated difference currents, whereas substitution of aspartate for Cl- had a greater effect on the outward than on the inward difference currents. The reversal potential in NaCycl-Ringer solution was estimated to be -32.5 mV, but this value is subject to considerable uncertainty given the very small values of the recorded difference currents.

Effects of ionic composition on swelling-activated Cl- channels. We used a protocol similar to that of Fig. 9A in examining the effects of external Cl-, NO3-, aspartate, and cyclamate on currents through swelling-activated Cl- channels in the same line of human NPE cells. As illustrated by Fig. 10A, the effects of partial replacement of external Cl- by aspartate or cyclamate were qualitatively similar to those noted for the IB-MECA-activated channels (Fig. 9A). Aspartate appeared to reduce the outward currents with little effect on the inward currents, whereas cyclamate reduced both outward and inward currents equally. Once again, this differential effect was suggested by using Eqs. 7 and 8 in estimating the scaling factors mapping the difference currents in NaAsp and NaCycl to those in NaCl (Table 2). The scaling factor for cyclamate was not significantly different in the hyper and depolarizing voltage ranges [(Delta kCycl)Swell = 0.2 ± 0.1], whereas the difference in scaling factors for aspartate was significant [(Delta kAsp )Swell = 0.27 ± 0.06; P < 0.01]. NPPB markedly inhibited both inward and outward currents.


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Fig. 10.   Effects of swelling as a function of the dominant external anions. A: the swelling-activated currents displayed a similar dependence on the dominant external anion. Partial substitution of external Cl- with I- was noted to have only a small effect (as seen here) and was not therefore systematically pursued in this study. The mean swelling-activated currents of 3 experiments were similar with NaCl- and NaI-Ringer solutions, but the corrected reversal potential was shifted negatively by 2.5 ± 0.9 mV by the I- substitution. B: difference currents for cells perfused with NaCl- and NaNO3-Ringer solutions, and NPPB difference currents for stimulated cells after cell swelling. C: difference currents for cells perfused with NaAsp- and NaCycl-Ringer solutions for cells activated by hypotonic perfusion.

The mean swelling-activated difference currents measured in the various perfusates are displayed in Fig. 10, B and C. As in the case of the IB-MECA-activated currents, the I-V relationships in NaCl- and NaNO3-Ringer solutions were similar to each other and to the NPPB difference currents obtained in NaCl-Ringer solutions. Once again, aspartate substitution for Cl- reduced the outward currents and shifted the reversal potential. The magnitudes of all the activated currents were larger in this series of experiments than those of the IB-MECA-activated currents of Fig. 9. The effects produced by partial replacement of external Cl- with cyclamate can therefore be more readily identified. As noted above, there was a marked reduction in both the outward and inward currents. In the case of Fig. 10C, the reversal potential in the NaCycl-Ringer solution can now be more clearly identified and is not significantly different from that of aspartate.

We used equations analogous to Eqs. 5 and 6 to estimate the relative permeabilities for Cl-, aspartate, and NO3- through the swelling-activated channels. With the use of the corrected reversal potentials entered in Table 4, we calculated values of 0.21 for (PAsp /PCl) and 0.87 for (PNO3/PCl).

                              
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Table 4.   Estimates of reversal potentials for swelling-activated currents

Figure 11 presents the time courses for representative difference currents in response to voltage pulses across the swelling-activated channels. Both the swelling-activated difference currents in different anions and the NPPB difference current in NaCl-Ringer solution are very similar to the corresponding traces for the IB-MECA-activated channels (Fig. 7). A prominent inactivation was noted at highly depolarizing voltages, and a variable small activation was noted at highly hyperpolarizing potentials.


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Fig. 11.   Swelling-activated and NPPB difference currents as functions of time following voltage steps. As in Fig. 6, swelling-activated difference currents were generated by subtracting the currents in the control period from the corresponding currents during the response to hypotonic perfusion with solution of the same ionic composition [NaCl (A), NaAsp (C), and NaCycl (D); see Table 2]. The NPPB difference currents (B) were measured as the NPPB-sensitive currents in hypotonic NaCl-Ringer solution.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are that the selective A3-subtype adenosine agonist IB-MECA activates Cl- channels and that the anionic dependence of these channels is similar to that of the swelling-activated Cl- channels of the same human line of nonpigmented ciliary epithelial cells.

We have previously reported that adenosine and nonspecific analogs activate Cl- channels of cultured human NPE cells (3), consistent with reports that A1-subtype adenosine receptors activate a 305-pS Cl- channel in RCCT-28A rabbit cortical collecting duct cells (29) and that Cl- channels are likely involved in adenosine-mediated effects in rat hippocampal slices (27). However, a previous report implicating A3-subtype adenosine receptors in the modulation of Cl- channels was based on indirect evidence from measurements of cell volume, short-circuit current, and RT-PCR amplification of RNA from cells and tissue (22). Use of highly selective agonists and antagonists permitted unambiguous demonstration that the observed effects of adenosine were mediated through A3 receptors, but the evidence that Cl- channels were indeed activated was less compelling. In the present study, we have observed that the A3-selective agonist IB-MECA activates Cl- channels with a dose dependence similar to the volume response, that similar currents are stimulated by the highly-selective agonist Cl-IB-MECA, that the reversal potential for the IB-MECA-stimulated currents is strongly dependent on the external Cl- concentration, that the currents can be inhibited by niflumic acid (500 µM) or by a low concentration (10 µM) of the Cl- channel blocker NPPB and essentially abolished by a higher concentration (100 µM), and that this higher concentration of NPPB also blocks the shrinkage induced by IB-MECA. It seems reasonable to conclude that A3-receptor specificity (established in Ref. 22) is mediated by activation of Cl- channels (documented in the current work).

From measurements of the reversal potential, we have calculated that the relative permeabilities through the IB-MECA-activated channels are PNO3 ~ PCl PAsp. We cannot estimate the relative permeability for cyclamate because its application to the external solution blocks both inward and outward currents by a mechanism yet to be identified. Perfusion with external cyclamate reduced the inward current conductance of both the IB-MECA- and swelling-activated currents by at least threefold (P < 0.001). This effect appears to be voltage independent (Fig. 9C) and could, therefore, reflect slow permeation through the pore or blocking action outside the conduit.

Single-channel patch-clamp measurements of bovine nonpigmented ciliary epithelial cells have identified at least two types of swelling-activated Cl- channels (38), and (in principle) other functionally heterogeneous Cl- channels could also be present. We had anticipated that perfusion with different anions [especially cyclamate (30)] might permit us to distinguish functionally between the IB-MECA-activated and swelling-activated Cl- channels. However, the relative permeabilities for Cl-, NO3-, and aspartate were similar for IB-MECA-activated and swelling-activated Cl- channels, and cyclamate produced a similar block in each. Small differences were indeed detected. For example, the relative permeability for NO3- was 1.03 for the adenosine-activated and 0.86 for the swelling-activated channels. However, these small differences can reflect the uncertainties of the techniques, including the necessarily approximate corrections for junction potential. The measured junction potentials agreed with the values calculated with the Henderson equation within 2.4 mV. If we take this value to represent the uncertainty of the reversal potential measurement, we calculate that a channel with a true PNO3/PCl ratio of 1 could display apparent values of 0.89 to 1.12, accounting for the range of values currently calculated.

We conclude that 1) the stimulation of A3 receptors by IB-MECA activates Cl- channels, and 2) IB-MECA-activated and swelling-activated Cl- channels of cultured human NPE cells are functionally similar in terms of their dependence on external Cl-, NO3-, aspartate, and cyclamate concentration, their inhibition by NPPB, and their inactivating characteristics following step changes in voltage. Previous studies on the short-circuit current across rabbit iris ciliary body have suggested that the Cl- conductance activated by adenosine (3) and IB-MECA (22) is present on the basolateral surface of the NPE cells facing the aqueous humor. Activation of these channels would thus permit efflux of Cl- out of the NPE cells and into the interior of the eye and a corresponding increase in the rate of aqueous humor production. The similarity between the IB-MECA-stimulated and the swelling-stimulated Cl- currents shown in the present study suggests that the latter could also be linked to an increase in aqueous humor production. We hypothesize that adenosine arises from the extracellular breakdown of ATP released from pigmented ciliary epithelial and NPE cells (21) and that substances that lead to ATP release, for example, those which elevate Ca2+, could lead to the stimulation of A3 receptors. The physiological trigger capable of activating the swelling-associated pathway remains to be elucidated.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Kenneth Jacobson (National Institutes of Health) for the MRS-1523. 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

Supported in part by National Eye Institute Research Grants EY-08343, EY-11213, and EY-01583 (for core facilities) and National Heart, Lung, and Blood Institute Respiratory Training Grant HL-07027 (to C. H. Mitchell).

Address for reprint requests and other correspondence: M. M. Civan, Dept. of Physiology, Univ. of Pennsylvania, A303 Richards Bldg., Philadelphia, PA 19104-6085.

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.

Received 17 November 1999; accepted in final form 7 March 2000.


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